Synthesis of ruthenium complexes functionalized with benzothiophene and their antibacterial activity againstStaphylococcus aureus

Article abstract of DOI:10.1039/d0dt04258g

New effective antimicrobial agents with novel modes of action are urgently needed due to the continued emergence of drug-resistant bacteria. Here, three ruthenium complexes functionalized with benzothiophene: [Ru(phen)₂(BTPIP)](ClO₄)

Full text of DOI:10.1039/d0dt04258g

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Synthesis of ruthenium complexes functionalized
with benzothiophene and their antibacterial
activity against Staphylococcus aureus†
Cite this: Dalton Trans., 2021, 50,
5607
a
b
c
a
Xiangwen Liao, *‡ Lianghong Liu,‡ Yanhui Tan,‡ Guijuan Jiang,*
a
a
a
d
Haihong Fang, Yanshi Xiong,
Xuemin Duan,
Guangbin Jiang
and
a
Jintao Wang*
New effective antimicrobial agents with novel modes of action are urgently needed due to the continued
emergence of drug-resistant bacteria. Here, three ruthenium complexes functionalized with benzothio-
2 4 2 2 4 2 2
phene: [Ru(phen) (BTPIP)](ClO ) (Ru(II)-1), [Ru(dmp) (BTPIP)](ClO ) (Ru(II)-2) and [Ru(dmb) (BTPIP)]
(
ClO (Ru(II)-3) (dmb = 4,4’-dimethyl-2,2’-bipyridine, phen = 1,10-phenanthroline, dmp = 2,9-
4 2
)
dimethyl-1,10-phenanthroline) have been synthesized and their antimicrobial activities in vitro were
assessed. Minimum inhibitory concentration (MIC) assays indicated that the three Ru(II)-1, Ru(II)-2 and
Ru(II)-3 complexes all showed antibacterial activity against Staphylococcus aureus and Pseudomonas
aeruginosa. The most active Ru(II)-3 complex was further tested against biofilms. Furthermore, it was also
tested whether complex Ru(II)-3 could serve as an antibacterial adjuvant. Interestingly, the checkerboard
data showed that Ru(II)-3 selectively exhibited synergism with aminoglycoside antibiotics. More impor-
tantly, the observed synergetic effect might be attributed to the inhibition of the regulatory function of
SaCcpA. Finally, in vivo bacterial infection treatment studies through a murine skin infection model and
skin irritation test were also conducted. All in all, these results confirmed that ruthenium complexes func-
tionalized with benzothiophene have good antimicrobial activity against Staphylococcus aureus.
Received 15th December 2020,
Accepted 20th March 2021
DOI: 10.1039/d0dt04258g
rsc.li/dalton
5
damaged skin surfaces. This pathogen can infect almost every
Introduction
tissue and produces various virulence factors in a human
6
Today, antibiotics have become the cornerstone of modern host. Worse still, resistance to vancomycin, linezolid and dap-
1
medicine. However, the continued emergence of drug-resist- tomycin has already been reported in clinical MRSA (methicil-
7
–9
ant bacteria, coupled with the decline in the discovery of new lin-resistant S. aureus) strains.
Obviously, there is clearly a
antimicrobial drugs in the pharmaceutical pipeline, has need for the development of new antimicrobials. More impor-
2
–4
created a public health crisis.
aureus) is the leading cause of both hospital and community agents with novel modes of action.
associated infections worldwide and a principal cause of mor- Compared to traditional organic antimicrobial drugs, metal
Staphylococcus aureus (S. tantly, there is a pressing need for new effective antimicrobial
1
0
bidity and mortality. It is worth noting that S. aureus is a com- complexes are promising in this regard as they offer alternative
mensal that colonizes the nares, axillae, vagina, pharynx, or electronic and stereochemical properties. It is worth noting
that the antimicrobial activity of transition metal complexes
1
1–16
has been widely tested in recent years.
For example, silver
a
School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang,
30013, China. E-mail: jgjchem@163.com, liao492008522@163.com
or copper(II)-based complexes were reported to show meaning-
1
6–18
3
ful antimicrobial activity.
Interestingly, polypyridyl ruthe-
b
School of Pharmaceutical Sciences, Hunan University of Medicine, Huaihua,
nium(II) complexes were reported to show significant bacteri-
4
18000, China
19
cidal activity against methicillin-resistant S. aureus strains.
c
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal
More importantly, ruthenium coordinated to established
Resources, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin,
41004, China
2
0
5
d
organic drugs could enhance their antimicrobial activities.
Guangxi Key Laboratory of Electrochemical and Magneto-chemical Function
Materials, College of Chemistry and Bioengineering, Guilin University of Technology,
Guilin, 541004, China
In a previous study, we designed and synthesized a series of
ruthenium polypyridyl complexes. The antibacterial activity
studies indicated that the ruthenium complexes formed by the
benzothiophene-substituted ligands showed better antibacter-
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0dt04258g
These author contribute equally to this work.
2
1
‡
ial activity.
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Scheme 1 The synthetic route of ligand and ruthenium complexes.
As ruthenium complexes formed by the benzothiophene- (15 mmol, 1156.2 mg) and acetic acid (30 mL) was refluxed
substituted ligands have strong antibacterial potential, we with stirring for 4 h. The cooled solution was diluted with
aimed to develop novel ruthenium complexes with better water and neutralized with concentrated aqueous ammonia.
activity based on a benzothiophene-substituted ligand. For The brown precipitate was collected and purified by column
this purpose, we designed and synthesized three ruthenium chromatography on silica gel (60–100 mesh) with ethanol as
complexes functionalized with benzothiophene: [Ru eluent to give the compound as a brown yellow powder. Yield:
(
(
phen) (BTPIP)] (ClO4)2 (Ru(II)-1), [Ru(dmp) (BTPIP)] (ClO )
Ru(II)-2) and [Ru(dmb) (BTPIP)](ClO ) (Ru(II)-3) (Scheme 1). 1588, 1480, 1358, 1312, 1188, 1121, 953, 819, 736, 682 cm ;
2 4 2
188.3 mg, 88%. IR: ν = 3101, 2102, 1916, 1793, 1691, 1597,
2
2
4 2
−
1
1
Their antimicrobial activities against S. aureus and
6
H NMR (400 MHz, DMSO-d ) δ 9.03 (d, J = 4.0 Hz, 2H), 8.94
P. aeruginosa were investigated. Subsequently, the Ru(II)-3 (d, J = 8.1 Hz, 2H), 8.40 (dd, J = 22.3, 8.3 Hz, 2H), 7.99 (d, J =
complex which exhibited the best bactericidal effect was 10.1 Hz, 5H), 7.88–7.80 (m, 3H), 7.40 (t, J = 6.8 Hz, 2H), 3.49 (s,
+
further tested against biofilms. In addition, in order to investi- 0H); HRMS (ESI) m/z: calcd for C27
H
17
N
4
S [M + H] , 429.1168;
gate whether ruthenium complexes could also serve as anti- found 429.1169.
biotic adjuvants, studies of the synergism between Ru(II)-3
Synthesis of [Ru(phen)
2 4 2
(BTPIP)](ClO ) (Ru(II)-1). A mixture
and common antibiotics against S. aureus were performed. of cis-[Ru(phen)
2
Cl ]·2H O (170.4 mg, 0.3 mmol) and BTPIP
2
2
Then the possible mechanism of the observed synergetic effect (128.4 mg, 0.3 mmol) in ethylene glycol (12 mL) was heated at
was investigated. Finally, the antimicrobial activities of Ru(II)- 150 °C under argon for 8 h to give a clear red solution. Upon
3
in vivo were investigated through a murine skin infection cooling, a red precipitate was obtained by dropwise addition of
model and a skin irritation test were also conducted to investi- saturated aqueous NaClO4 solution. The crude product was
gate the irritation effect of Ru(II)-3 on the skin.
purified by column chromatography on neutral alumina with a
mixture of CH CN–toluene (1 : 1, v/v) as eluent. The red band
3
was collected. The solvent was removed under reduced
pressure and a red powder was obtained. Yield: 254.8 mg,
Experimental
Chemistry
7
1
8%. IR: ν = 3051, 1601, 1576, 1508, 1478, 1455, 1426, 1363,
304, 1250, 1224, 944, 843, 808, 750, 623, 528 cm ; H NMR
) δ 9.13 (dd, J = 24.7, 7.9 Hz, 1H), 8.90 (dd,
−1 1
Synthesis of ligand (BTPIP). A mixture of 1,10-phenanthro- (400 MHz, DMSO-d
6
line-5,6-dione (100.00 mg, 0.5 mmol), 4-(benzo[b]thiophen-2- J = 14.6, 8.1 Hz, 2H), 8.47 (dd, J = 41.7, 7.2 Hz, 1H), 8.24 (t, J =
yl)benzaldehyde (119.0 mg, 0.5 mmol), ammonium acetate 7.8 Hz, 2H), 8.13 (t, J = 7.7 Hz, 2H), 8.00 (t, J = 9.7 Hz, 4H),
5608 | Dalton Trans., 2021, 50, 5607–5616
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7
.96–7.81 (m, 7H), 7.70–7.61 (m, 4H), 7.41 (d, J = 6.3 Hz, 4H); DMSO was tested in the same way and the solvent showed no
1
3
C NMR (100 MHz, DMSO-d ) δ 157.3, 157.1, 151.9, 149.1, antimicrobial activity.
6
1
1
1
49.2, 149.3, 145.2, 145.1, 143.1, 141.0, 139.2, 138.4, 138.2,
34.5, 130.9, 128.4, 128.2, 127.6, 127.7, 126.8, 126.2, 126.3, Overnight cultured S. aureus strains were 1 : 100 diluted with
Ruthenium complexes inhibited the growth of S. aureus.
25.4, 125.5, 124.9, 125.0, 124.8, 124.4, 123.0, 121.0, 115.4; fresh medium and then further cultured at 37 °C.
+
HRMS (ESI) m/z: calcd for C51
89.1443; found 889.1451.
H
31
N
8
SRu [M − 2ClO
4
− H] , Subsequently, the logarithmic phase bacteria were 1 : 10
diluted again. After the addition of different concentrations of
8
Synthesis of [Ru(dmp) (BTPIP)](ClO ) (Ru(II)-2). This ruthenium complexes, the bacteria were cultured in a shaker
2
4 2
complex was synthesized in an identical manner to that at 37 °C. Then the OD600 of the bacteria were measured every
described for complex [Ru(phen) (BTPIP)](ClO , with cis-[Ru 30 minutes.
dmp) Cl ]·2H O in place of cis-[Ru(phen) Cl ]·2H O. Yield: Thiol detection in S. aureus. At first, different concen-
37.1 mg, 69%. IR: ν = 3053, 1964, 1605, 1588, 1507, 1478, trations of Ru(II)-3 were added into the logarithmic phase bac-
2
4 2
)
(
2
2
2
2
2
2
2
1
433, 1348, 1248, 1216, 1089, 854, 809, 726, 622, 555 cm− ; H teria. After further culturing for 30 min, the bacteria were col-
1
1
NMR (400 MHz, DMSO-d ) δ 8.93 (d, J = 8.3 Hz, 2H), 8.83 (d, lected by centrifugation and washed twice with PBS. Finally, a
6
J = 8.0 Hz, 2H), 8.48–8.43 (m, 4H), 8.28 (t, J = 10.2 Hz, 4H), Thiol Detection Assay Kit (Cayman) was used to determine the
8
7
1
1
1
1
1
2
.02–7.96 (m, 4H), 7.94–7.87 (m, 3H), 7.52–7.44 (m, 2H), concentration of the free thiol in S. aureus.
.43–7.34 (m, 6H), 7.19 (m, J = 34.5, 7.6 Hz, 1H), 1.97 (s, 6H), The effect of Ru(II)-3 on biofilm formation. The previous
) δ 168.4, 166.8, day, 1 mL of 10% albumin from bovine serum were added into
50.7, 150.3, 149.4, 149.0, 148.3, 145.9, 143.6, 142.9, 140.9, 24-well microtiter plates and stored at 4 °C. The albumin from
39.2, 138.6, 137.8, 137.2, 135.0, 131.0, 130.8, 120.0, 129.4, bovine serum was removed from the plate before use.
28.7, 128.0, 127.9, 127.6, 127.0, 126.3, 125.8, 125.4, 124.4, Overnight cultured S. aureus strains were 1 : 100 diluted into
1
3
.76 (s, 6H); C NMR (100 MHz, DMSO-d
6
23.0, 121.3; HRMS (ESI) m/z: calcd for C55
H
39
N
8
SRu [M − fresh TSB medium. After further culturing for 5 h, the bacteria
+
ClO − H] , 945.2070; found 945.2088.
were diluted 1 : 200 into TSB medium containing 0.5% glucose
4
Synthesis of [Ru(dmb)
2
(BTPIP)](ClO
4
)
2
(Ru(II)-3). This and different concentrations of Ru(II)-3. Subsequently, 1 mL
complex was synthesized in an identical manner to that aliquots of the bacterial suspension were added into the
described for complex [Ru(phen) (BTPIP)](ClO ) , with cis- 24-well plate. After culturing at 37 °C for 36 h, the bacterial
2
4 2
[Ru(dmb)
2
Cl
2
]·2H
2
O in place of cis-[Ru(phen)
2
Cl
2
2
]·2H O. Yield: suspension was removed and the plate was washed with PBS
2
66.6 mg, 81%. IR: ν = 3057, 2919, 1618, 1507, 1479, 1448, three times. The adherent bacteria were dried out overnight at
−
1 1
1
364, 1304, 1197, 1093, 953, 825, 725, 566, 544 cm ; H NMR room temperature and were then strained through 0.1% crystal
) δ 9.06 (dd, J = 13.5, 8.2 Hz, 2H), 8.75 (d, violet solution (Sangon, China). The crystal violet solution was
J = 15.5 Hz, 4H), 8.39 (dd, J = 22.9, 7.7 Hz, 2H), 7.98 (m, J = removed and the plate washed with PBS again after
1.6, 22.2, 5.8 Hz, 10H), 7.70 (s, 2H), 7.48–7.36 (m, 6H), 7.20 15 minutes. The biofilm formation could be determined
(400 MHz, DMSO-d
6
3
1
3
(
d
1
1
1
s, 2H), 2.58 (s, 6H), 2.48 (s, 6H); C NMR (100 MHz, DMSO- through monitoring the absorbance at 595 nm after the
) δ 156.8, 156.7, 153.6, 151.0, 150.9, 150.0, 149.9, 149.8, addition of 1 mL of acetic acid.
45.6, 142.9, 140.9, 139.3, 135.1, 135.0, 130.7, 130.5, 129.4, The synergism study between Ru(II)-3 and antibiotics. At
29.0, 128.8, 127.7, 127.0, 127.0, 126.5, 125.5, 125.4, 124.4, first, the MIC values of all selected antibiotics against S. aureus
6
2
2
22.96, 121.3, 121.0, 21.2, 21.1; HRMS (ESI) m/z: calcd for Newman were examined, as described previously. Next, over-
+
C H N SRu [M − 2ClO − H] , 897.2069; found 897.2077.
night cultured S. aureus were 1 : 100 diluted into fresh TSB
medium and further incubated until the OD600 reached 1. The
bacteria were 1 : 20 diluted into fresh TSB medium to obtain a
bacterial suspension. Subsequently, 200 μL of bacterial sus-
5
1
39
8
4
Biology
Bactericidal activity. The bacterial strains were purchased pension with a gradient concentration of Ru(II)-3 and anti-
from China Center of Industrial Culture Collection (CICC). The biotics were transferred into the wells of 96-well plates. After
frozen bacteria at −80 °C were scratched on a TSB Agar plate. incubation for 20 h at 37 °C, the fractional inhibitory concen-
After growing at 37 °C overnight, a single colony was picked tration index (FICI) was calculated. GraphPad Prism software
from the TSB Agar plate and cultured in TSB medium. was employed for plotting isobolograms.
Minimal inhibitory concentrations (MICs) of ruthenium com-
plexes against S. aureus (ATCC 25904) and P. aeruginosa (ATCC gene. S. aureus Newman were cultured in TSB medium the
027) were measured. In brief, the overnight cultured bacteria night before. Then the overnight bacteria were 1 : 100 diluted
Real-time PCR investigation of the transcription level of
9
were diluted into fresh TSB medium. After culturing for into fresh TSB and cultured at 37 °C. After the bacteria had
another 2 hours, the bacteria were 1 : 100 diluted with fresh grown into a logarithmic phase, different concentrations of
medium to get a bacterial suspension. The MIC assays were Ru(II)-3 were added. S. aureus was collected by centrifugation
performed with a 96-well plate. After the addition of 200 µL of after another 1 hour’s incubation at 37 °C. The SV total RNA
bacterial suspension with different concentrations of ruthe- isolation system (Promega) was used for total RNA extraction.
nium complexes, the plate was incubated at 37 °C. The anti- Subsequently, a GoTaq qPCR Master Mix kit (Promega) was
microbial activity of the solvent with the same amount of employed to investigate the transcription level of the gene
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through real-time PCR (Life Technologies). Relative transcrip- Table 1 Minimum inhibition concentration (MIC) of all the compounds
against S. aureus Newman bacterial strains
−
ΔΔ
tion levels were calculated using the
6S rRNA as an internal control. The values of the untreated
group were set as 1. The Ru(II)-3 treated groups were normal-
Ct method by setting
1
Compounds
Minimum inhibition concentration (MIC)
1
1
ized to that of control groups. The primers used were: 16S Ru(II)-1
0.050 mg mL−
Ru(II)-2
rRNA-For and 16S rRNA-Rev, pckA-For and pckA-Rev, ccpA-For
Ru(II)-3
0.025 mg mL−
−1
0.003 mg mL−
1
and ccpA-Rev, citZ-For and citZ-Rev.
Gentamicin
0.005 mg mL
>0.250 mg mL−
1
Investigation of the antibacterial activity in vivo. First, hair RuCl
removal cream (Veet®) was smeared on the infection site of
female BALB/c mice (6–8 weeks of age, 20–25 g in weight) to
3
·3H O
2
remove the hair one day before infection. Subsequently, over- activity. This result indicated that 2,2′-bipyridine ligands are
night cultured S. aureus Newman strains were 1 : 100 diluted in more beneficial to enhancing the antibacterial activity of
fresh TSB medium and grown at 37 °C until OD600 reached 0.6. ruthenium complexes. While [Ru(dmb) (BTPIP)] (ClO ) (Ru
2 4 2
The bacteria were harvested by centrifugation and the cell (II)-3) formed from 4,4′-dimethyl-2,2′-bipyridine showed the
pellet was washed with PBS three times for further use. The best bactericidal effect. Given that Ru(II)-2 formed from 2,9-
abscesses on the mice were developed through subcutaneous dimethyl-1,10-phenanthroline also showed lower MIC com-
7
injection of 50 μL of S. aureus Newman (3 × 10 CFU) which pared with Ru(II)-1, it is rational to believe that ruthenium
was resuspended with PBS. All the mice were divided into complexes with better activity could be acquired by increasing
three groups (n = 5 for each group) including a control and a the lipophilicity of auxiliary ligands.
−1
−1
treatment group (Ru(II)-3, 0.05 mg mL or 0.10 mg mL ).
Furthermore, the effects of ruthenium complexes on
Before treatment, sterile cream was prepared by mixing stearic S. aureus growth were measured by growth curves. As shown in
acid (4.8 g), glycerin monostearate (1.4 g), liquid paraffin Fig. 1, the inhibitory effects of the three ruthenium complexes
(
(
2.4 g), albolene (0.4 g), lanum (2.0 g), and distilled water on S. aureus were dose-dependent. The growth of S. aureus was
29 g). Subsequently, the mice received twice-daily treatment remarkably inhibited in the presence of the three ruthenium
through cream containing different concentrations of Ru(II)-3 complexes (Ru(II)-1, Ru(II)-2, Ru(II)-3). Once again, there was
smeared onto the abscesses. After 64 hours, all the mice were scarcely any bacterial growth observed after incubation with
sacrificed and the abscess tissues were collected. Tissue homo- the three ruthenium complexes at their minimum inhibitory
genates were serially diluted in PBS and then spread onto agar concentration (MIC).
plates for the enumeration of bacterial load.
Next, the antibacterial activities of the three ruthenium
Acute dermal irritation test. Female BALB/c mice were ran- complexes against Gram-negative pathogenic bacterium
domly divided into three groups: Control, Ru(II)-3 (0.05 mg (Pseudomonas aeruginosa) were also investigated. The results
−
1
−1
mL ) and Ru(II)-3 (0.10 mg mL ). The hair coat of the dorsal indicated that the three compounds (Ru(II)-1, Ru(II)-2, Ru(II)-
area from female mice was clipped and removed on the day 3) also showed antibacterial activity against Pseudomonas aeru-
−
1
before the test. Compounds and a distilled water control were ginosa with MIC ranging from 0.15 to 0.25 mg mL
softly attached to the shaved part (about 2 cm ) once a day for (Table S1†).
2
three days. On the fourth day the mice were killed by cervical
Many metal drugs which show significant antibacterial
dislocation and their shaved parts were removed and fixed in activity have been reported that could decrease cellular free
2
3,24
4
% paraformaldehyde at 4 °C for 1 day, then embedded in thiols via ROS production.
Subsequently, a thiol detection
paraffin. Sequential sections were prepared for HE assays.
assay kit was employed to determine intracellular free thiols
level in S. aureus after Ru(II)-3 treatment. The results suggested
that Ru(II)-3 treatment could decrease cellular free thiols in
S. aureus and the mode of action of the compounds might
include increased generation of intracellular reactive oxygen
species (ROS) (Fig. S1†).
Results and discussion
The antibacterial activity studies
First of all, we evaluated the antibacterial activity of three
ruthenium complexes functionalized with benzothiophene
Inhibition of biofilm formation
against S. aureus (Newman) using minimum inhibitory con- It is well known that antibiotic resistance of bacteria is partly
centration (MIC) assays. From the results in Table 1, as we related to virulence factor secretion and biofilm formation.
expected, the three compounds all showed good antibacterial Bacteria could maintain stable growth in stressful environ-
activity against S. aureus, with MIC ranging from 0.003 to mental conditions through biofilm production. In fact, the
.050 mg mL−
1
.
Interestingly, [Ru(dmb)
(BTPIP)] (ClO
)
biofilm mainly consists of exopolysaccharides (EPS), proteins
0
(
2
4
2
2
5
Ru(II)-3) showed the best antibacterial effect against S. aureus. and DNA and then adheres to abiotic surfaces. In order to
Compared with ruthenium complexes formed by 2,2′-bipyri- further investigate the antibacterial activity, the most active
dine which we previously reported ([Ru(bpy) (BTPIP)](ClO Ru(II)-3 complex was further tested against biofilm formation.
(BTPIP)] (ClO (Ru(II)-1) The drug concentration for biofilm formation assays was a
formed with 1,10-phenanthroline showed weaker antibacterial sublethal concentration to ensure the inhibition of biofilm for-
2
4 2
) ,
MIC = 0.016 mg mL− ), [Ru(phen)
1
2
4 2
)
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Fig. 1 The growth curve of S. aureus Newman strains in the presence of ruthenium complexes. The OD600 was recorded at 30 min intervals.
A) Ru(II)-1, (B) Ru(II)-2, (C) Ru(II)-3.
(
2
0
mation was not due to killing of the bacteria. In brief, after described. All in all, we selected four commonly used anti-
adding different concentrations of Ru(II)-3 complex, S. aureus biotics: ampicillin (β-lactam antibiotics); levofloxacin (quino-
were transferred into a 24-well plate and further incubated at lone antibiotic); gentamicin (aminoglycoside antibiotics); and
3
7 °C for 36 h. The biofilm formation could be determined chloramphenicol. Firstly, S. aureus Newman bacterial suspen-
6
−1
through crystal violet staining. As shown in Fig. 2, in the pres- sion (5 × 10 CFU mL ) with gradient concentrations of
−
1
ence of 0.001 or 0.002 mg mL of Ru(II)-3, the biofilm for- Ru(II)-3 and selected antibiotics were added into each well of
mation was significantly reduced by 10.5 and 25%, respect- the 96-well plate. After incubation at 37 °C for 20 hours, the
ively. This indicated that ruthenium complexes could signifi- FICI values were calculated. The definition of FICI (fractional
cantly reduce biofilm formation at sublethal concentration.
inhibitory concentration index) is the sum of the MIC values
of each drug when used in combination divided by the MIC
values of the drug when used alone. The synergy activity was
defined by FICI values ≤0.5 while the antagonism was defined
by FICI >4. As summarized in Fig. 3, the FICI values of the
combination with Ru(II)-3 and gentamicin was 0.187. This
data indicated that Ru(II)-3 exhibited synergism with gentami-
cin and Ru(II)-3 might increase antibacterial activity of some
aminoglycoside antibiotics against S. aureus.
Ruthenium complexes showed synergism with the
aminoglycoside antibiotics
The use of adjuvants is identified as an effective way to deal
with bacterial resistance. As we had found that ruthenium
complexes showed significant activity against Staphylococcus
aureus, we wondered whether ruthenium complexes could also
serve as adjuvants to enhance the action of existing antibiotics
against Staphylococcus aureus. Therefore, the checkerboard
method was performed to investigate the synergism between
Ru(II)-3 and common antibiotics against S. aureus as
Real-time qPCR
The checkerboard method confirmed that Ru(II)-3 exhibited
synergism with gentamicin against S. aureus. However, the
mechanism of the observed synergetic effect is unknown. In
our previous study, we found that bis(4-hydroxy-3-methyl-
phenyl) sulfide (HMS) which could abrogate the regulatory
function of SaCcpA (catabolite control protein
A from
Staphylococcus aureus) also selectively increased the suscepti-
2
6
bility of S. aureus to some aminoglycoside antibiotics.
Therefore, we speculated that the catabolite control protein A
SaCcpA) might also serve as one target of Ru(II)-3. For further
(
confirmation, real-time PCR was employed to investigate the
transcription of the gene which CcpA regulated after Ru(II)-3
treatment. As SaCcpA mainly binds to the catabolite-respon-
sive element (cre) sequences, we investigated the transcription
of citZ, hla and pckA in the S. aureus strain after supplemen-
tation with Ru(II)-3. As shown in Fig. 4, the transcription levels
of CcpA regulated genes (citZ, ccpA and pckA) were attenuated
by Ru(II)-3. This phenomenon confirmed that Ru(II)-3
restrained the gene regulatory activity of catabolite control
protein A (CcpA) in S. aureus.
Fig. 2 The effect of Ru(II)-3 on biofilm formation of S. aureus Newman
strains. All experiments were performed with three biological replicates.
The mean value of control groups is set as 100%.
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Fig. 3 The synergism between Ru(II)-3 and four antibiotics was investigated by checkerboard assay (A). (B) The MIC values of four common anti-
biotics and the FICI values of the combination with Ru(II)-3 against S. aureus. (C) The synergistic effects of Ru(II)-3 with antibiotics were analysed by
isobologram. (D) Heat plots of checkerboard assays for Ru(II)-3 in combination with four antibiotics against S. aureus Newman.
Synergism activity investigated against ccpA mutant strains
disturbed. For further confirmation, we performed the check-
erboard method again to investigate the synergism between
The real-time qPCR data showed that Ru(II)-3 exhibited selecti- Ru(II)-3 and aminoglycoside antibiotics against S. aureus
vity synergism with the aminoglycoside antibiotics which ΔccpA mutant (ccpA gene knockout). As we expected, the syner-
might be related to the regulatory function of SaCcpA being gism between Ru(II)-3 and aminoglycoside was completely
5612 | Dalton Trans., 2021, 50, 5607–5616
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SaCcpA. This also further accounts for the phenomenon that
Ru(II)-3 increased the susceptibility of S. aureus to aminoglyco-
side antibiotics.
In vivo bacterial infection treatment studies with mouse
The minimum inhibitory concentration assays and checker-
board method data all confirmed that ruthenium complexes
functionalized with benzothiophene have good antimicrobial
activity against Staphylococcus aureus. However, the in vivo anti-
bacterial activity of ruthenium complexes is unknown. It has
already been reported that ruthenium complexes would be
rapidly cleared from the bloodstream after administration, so
ruthenium complexes need to be suitable for topical appli-
cation for surface infection treatment rather than injection
2
7
routes. Therefore, a mouse skin infection model was carried
Fig. 4 The effect of Ru(II)-3 on transcription of pckA, ccpA and citZ in out in order to further confirm whether ruthenium complexes
S. aureus. All experiments were performed with three biological repli-
cates. The mean value of control groups is set as 1. Results are shown as
mice were inoculated with S. aureus Newman strains to
mean ± sd.
also possessed significant in vivo antibacterial activity. In brief,
develop abscesses on the skin. Subsequently, all the mice were
divided into three groups and then the ointment containing
Ru(II)-3 (50 μg mL− or 100 μg mL ) was smeared onto the
abolished in the S. aureus ΔccpA mutant (Fig. 5). Taken abscesses twice daily. After treatment for 64 hours, the abscess
together, our results clearly demonstrated that Ru(II)-3 exhibi- tissue was excised and homogenized for CFU quantification.
ted selectivity synergism with the aminoglycoside antibiotics The results are shown in Fig. 6, after treatment with Ru(II)-3
which might be by inhibiting the regulatory function of ointment (100 μg mL− ), smaller cutaneous abscesses were
1
−1
1
Fig. 5 Heat plots of checkerboard assays for Ru(II)-3 in combination with different antibiotics against S. aureus ΔccpA mutant strains.
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Fig. 6 The antibacterial activity of Ru(II)-3 in vivo was studied with a murine skin infection model (A). (B) The bacterial load of local abscesses
induced by S. aureus Newman was enumerated in control or Ru(II)-3 treatment groups. The logCFU values are presented as the mean ± sd. The stat-
istical difference is determined by a Mann–Whitney U test. (C and D) Representative photographs of the cutaneous abscesses in the presence/
absence of Ru(II)-3 therapy.
observed compared with the control group. In addition, the Skin irritation test in mice
viable bacterial counts dropped significantly compared to the
control group, with logCFU mean values of 9.8 (control) and Ru(II)-3 showed potential application for surface infection
−1
8
.2 (Ru(II)-3, 100 μg mL ). This phenomenon suggested that treatment. Finally, to investigate the irritation effect of Ru(II)-3
Ru(II)-3 indeed showed potential application for surface infec- on the skin, the skin reaction was measured. In brief, oint-
tion treatment.
ment containing Ru(II)-3 or distilled water control were softly
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Fig. 7 The irritation effect of Ru(II)-3 on the skin. (A) Control, 50 μg mL− (B) or 100 μg mL−1 (C) of Ru(II)-3. Representative histological results
1
(
hematoxylin and eosin (HE) stain of skin (Scale bar = 500 μm) are shown 4 days after attachment.
attached to the shaved part once a day for three days. On the This will prompt us to design better organic ligands to improve
fourth day the mice were killed by cervical dislocation and the antibacterial activity of ruthenium complexes in our next
their shaved parts were removed and fixed in 4% paraformal- work. In addition, the synergy observed in our study provides an
dehyde at 4 °C for 1 day, then embedded in paraffin. interesting starting point for further investigations.
Sequential sections were prepared for HE assays. As shown in
Fig. 7, there were no appreciable skin reactions including the
formation of erythema or edema during the experimental
period, HE stain also confirmed this. Thus, Ru(II)-3 could be
Ethics statement
considered to be a non-irritant material.
The animal study was performed in strict accordance with the
NIH guidelines for the care and use of laboratory animals
(
NIH Publication No. 85-23 Rev. 1985) and was reviewed and
approved by Institutional Animal Care and Use Committee of
Guangxi Normal University (Guilin, China).
Conclusions
There is clearly a need for the development of new classes of
antimicrobials to fight multidrug-resistant bacteria. In this
study, three ruthenium complexes functionalized with ben-
zothiophene were synthesized and their antimicrobial activities
Conflicts of interest
were assessed. Minimum inhibitory concentration (MIC) assays The authors have declared that there is no conflict of interest.
indicated that three of the ruthenium complexes (Ru(II)-1,
Ru(II)-2 and Ru(II)-3) all showed antibacterial activity against
S. aureus and P. aeruginosa. The most active Ru(II)-3 complex was
also tested against biofilm formation and could significantly
Acknowledgements
reduce biofilm formation at sublethal concentration. Ru(II)-3 We gratefully acknowledge the generous support provided by The
treatment could also decrease cellular free thiol in S. aureus. Natural Science Foundation of Jiangxi, China (20192BAB213002,
More importantly, a checkerboard assay indicated that Ru(II)-3 20192BAB20510); Department Education Science and Technology
also has potential as an antibiotic adjuvant because of Ru(II)-3 Research Project of Jiangxi, China (GJJ201139, GJJ180624); The
could increase the antibacterial activity of some aminoglycoside Open Project of Jiangxi Provincial Key Laboratory of Drug Design
antibiotics against S. aureus. In addition, the mechanism study and Evaluation (JKLDE-KF-KFGJ18017); Jiangxi Science
clearly demonstrated that Ru(II)-3 exhibited selectivity synergism Technology Normal University (2020XJYB018).
with the aminoglycoside antibiotics which might be by inhibit-
&
ing the regulatory function of SaCcpA. Finally, in vivo bacterial
infection treatment studies through a murine skin infection
model and skin irritation test were also conducted. The results
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