Antibacterial activity and mechanism of sanguinarine against Staphylococcus aureus by interfering with the permeability of the cell wall and membrane and inducing bacterial ROS production
Staphylococcus aureus (SA) is representative of
gram-positive bacteria. Sanguinarine chloride hydrate (SGCH) is the
hydrochloride form of sanguinarine (SG), one of the main extracts of Macleaya
cordata (M. cordata). There are few reports on its antibacterial mechanism
against SA. Therefore, in this study, we investigated the in vitro antibacterial
activity and mechanism of SGCH against SA. The inhibitory zone, minimum
inhibitory concentration (MIC), and minimum bactericidal concentration (MBC)
were measured, and the bactericidal activity curve was plotted. In addition,
the micromorphology, alkaline phosphatase (AKP) activity, NaK, Ca++2+Mg2+-adenosine
triphosphate (ATP) activity, intracellular reactive oxygen species (ROS), and
fluorescein diacetate (FDA) were observed and detected. The results showed that
the inhibitory zone of SGCH against SA was judged as
medium-sensitive; the MIC and MBC were 128 and 256 μg/mL, respectively; in the
bactericidal activity curve, SGCH with 8 × MIC could completely kill SA within
24 h. SGCH was able to interfere with the integrity and permeability of
the SA cell wall and membrane, as confirmed by the scanning electron
microscopy (SEM) images, the increase in extracellular AKP and Na K, Ca++2+ Mg2+-ATP
activities as well as the fluorescein diacetate (FDA) staining experiment
results. Moreover, a high concentration of SGCH could induce SA to
produce large amounts of ROS.
With the intensive and large-scale development of animal
husbandry in China, the incidence of bacterial diseases is also
increasing. SA is one of the major pathogens causing bacterial
infectious diseases. As early as 1999, Navarro studied the antibacterial effect
of Bocconia arborea and found that dihydrochelerythrine and dihydrosanguinarine
had the ability to inhibit the growth of gram-positive bacteria, with a
stronger inhibitory effect on Diplococcus pneumoniae, SA, and Bacillus
subtilis than berberine . Liang et
al. and Miao et al. both
found that SG compounds showed significant inhibition on SA and Escherichia
coli. In this study, the in vitro antibacterial activity
results showed that SGCH had a good inhibitory effect on SA, and
SGCH (1,000 μg/mL) had a higher antibacterial activity, which were consistent
with the results of these studies.
Many plant extracts play an antibacterial role by destroying the
cell microstructure. Ren et al. observed
the effect of aureusidin on the SA micromorphology, and the
results showed that the SA cells in the control group had a
complete structure, but the group treated by aureusidin was partially dissolved
and the cell surface became rough. Our results of SEM images showed that SA in
the control group was round and full with a smooth surface. SA treated
with SGCH showed collapse, surface dissolution, mutual adhesion, and leakage of
cell sap, indicating that SGCH could obviously destroy the micromorphology and
microstructure of SA cells.
The bacterial cell wall has the functions of protecting
bacteria, transporting substances and participating in the pathogenic process.
The damage of cell wall will affect its growth and the ability to resist the
external environment. AKP is located between the cell wall and the cell
membrane, which can be used as an indicator to detect the integrity of cell
wall .
In this study, the 8 × MIC group could induce the leakage of intracellular AKP,
thus increasing the activity of extracellular AKP, which indicated that a high
concentration of SGCH could effectively disrupt the cell wall of SA.
He et al. investigated the in vitro antibacterial mechanism of
chelerythrine against SA and found that the extracellular AKP
activity of bacteria in the experimental group was significantly increased,
which was consistent with the results of this study.
The damage of cell membrane will cause the leakage of cytoplasm,
which will seriously affect the metabolism of the bacterium. ATP exists on the
tissue and organelle membranes and is a protease on the biofilm. It plays an
important role in material transport, energy conversion, and information
transmission. The determination of ATP activity values can be used as an
indicator of the integrity of the cell membrane . In this
experiment, the high SGCH concentration increased the permeability of Na, Kand
Ca++2+, Mg2+, thus increasing the extracellular
Na K, Ca++2+ Mg2+-ATP activities in a
concentration-dependent manner, indicating that the high SGCH concentration can
effectively destroy the integrity of the SA cell membrane. Tao
et al. measured the intracellular Ca2+ Mg2+-ATP activity of
bacteria, which was significantly decreased in the experimental group, and the
trend was consistent with that of this study.
After FDA is hydrolyzed by non-specific lipase in cells, it will
produce fluorescein that can emit yellow-green fluorescence, and fluorescein
will be detected at excitation and emission wavelengths of 297 nm and 527 nm,
respectively. When the cell membrane is complete, fluorescein is present in the
cell, and high-intensity fluorescence can be detected. When the cell membrane
is disrupted, fluorescein will flow out of the cell quickly, and the
intracellular fluorescence intensity will be greatly decreased. The
permeability of the cell membrane can be reflected by the FDA fluorescence
intensity values . The
results of the FDA staining experiment in this study showed that different
concentrations of SGCH all caused the loss of fluorescein from the
intracellular bacteria, thus decreasing the fluorescence intensity of FDA,
which indicated that SGCH at different concentrations could significantly
damage the permeability of the SA cell membrane.
As an important signaling molecule, ROS can reflect the cellular
activity state. Although cells can continuously produce low levels of ROS to
maintain a normal cellular life activity, when the amount of intracellular ROS
exceeds the capacity of the antioxidant mechanism, excessive oxidative stress
will lead to irreversible damage to intracellular macromolecules . Tang et
al. explored the in vitro antibacterial mechanism of biogenic
tellurium nanoparticles and precursor tellurite against Escherichia
coli, and the measurement of ROS levels were found to be significantly
higher in the experimental group. In this study, the fluorescence intensity,
fluorescence spectroscopy, and CLEM results all reflected that different
concentrations of SGCH could induce SA to produce a large
amount of ROS, thus causing bacterial oxidative damage, which were consistent
with this study.
SA is a representative pathogen causing a variety of diseases
in humans and animals. It is sensitive to many antibiotics but also prone to
drug resistance. In the context of global antibacterial restriction, there is
an urgent need for safe and efficient alternative products in animal
production, the development of new sterilization methods and the reduction of
the use of antibiotics, so as to effectively control and prevent SA infections.
Natural plant extracts are ideal alternatives to antibiotics in animal
production because of their natural, multifunctional, low toxicity, high
safety, and non-resistant characteristics. SG is the main active ingredient of
M. cordata plants, and it has the advantages of fast absorption, rapid
distribution, fast metabolism, low bioavailability, and low body residue in the
organism, and will have excellent effects in various types of livestock and
poultry breeding. Our results revealed that SGCH has a preferable antibacterial
effect on SA so that SGCH could destroy the cellular structure
of SA, interfere with the permeability of the SA cell
wall and membrane, and induce oxidative damage in SA; thus, SGCH
has the potential to be exploited as an antibiotic substitution in animal
husbandry and for the clinical control and treatment of diseases caused
by SA.
In conclusion, SGCH has good antibacterial activity
against SA; Moreover, SGCH exerts its antibacterial mechanism by
destroying the cellular structure, interfering with the permeability and
integrity of the cell walls and membranes, and inducing SA oxidative
damage. SGCH may thus be a potential antimicrobial agent for control and
treatment of SA infections in the future research.