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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.

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