Impact of cell-free supernatant of lactic acid bacteria on Staphylococcus aureus biofilm and its metabolites.

Introduction: A safe bio-preservative agent, lactic acid bacteria (LAB) can inhibit the growth of pathogenic bacteria and spoilage organisms. Its cell-free supernatant (LAB-CFS), which is rich in bioactive compounds, is what makes LAB antibacterial work. Methods: This study focused on the changes in biofilm activity and related metabolic pathways of S. aureus treated with lactic acid bacteria planktonic CFS (LAB-pk-CFS) and biofilm state (LAB-bf-CFS). Results: The findings demonstrated that the LAB-CFS treatment considerably slowed Staphylococcus aureus (S. aureus) growth and prevented it from forming biofilms. Additionally, it inhibits the physiological traits of the S. aureus biofilm, including hydrophobicity, motility, eDNA, and PIA associated to the biofilm. The metabolites of S. aureus biofilm treated with LAB-CFS were greater in the LAB-bf-CFS than they were in the LAB-pk-CFS, according to metabolomics studies. Important metabolic pathways such amino acids and carbohydrates metabolism were among the most noticeably altered metabolic pathways. Discussion: These findings show that LAB-CFS has a strong potential to combat S. aureus infections.

Baicalein Inhibits the Staphylococcus aureus Biofilm and the LuxS/AI-2 System in vitro.

Introduction: Staphylococcus aureus (S. aureus) is a common cause of mastitis in dairy cows, a condition that has a significant economic impact. S. aureus displays quorum sensing (QS) system-controlled virulence characteristics, like biofilm formation, that make therapy challenging. In order to effectively combat S. aureus, one potential technique is to interfere with quorum sensing. Methods: This study evaluated the effects of different Baicalin (BAI) concentrations on the growth and the biofilm of S. aureus isolates, including the biofilm formation and mature biofilm clearance. The binding activity of BAI to LuxS was verified by molecular docking and kinetic simulations. The secondary structure of LuxS in the formulations was characterized using fluorescence quenching and Fourier transform infrared (FTIR) spectroscopy. Additionally, using fluorescence quantitative PCR, the impact of BAI on the transcript levels of the luxS and biofilm-related genes was investigated. The impact of BAI on LuxS at the level of protein expression was also confirmed by a Western blotting investigation. Results: According to the docking experiments, they were able to engage with the amino acid residues in LuxS and BAI through hydrogen bonding. The results of molecular dynamics simulations and the binding free energy also confirmed the stability of the complex and supported the experimental results. BAI showed weak inhibitory activity against S. aureus but significantly reduced biofilm formation and disrupted mature biofilms. BAI also downregulated luxS and biofilm-associated genes' mRNA expression. Successful binding was confirmed using fluorescence quenching and FTIR. Discussion: We thus report that BAI inhibits the S. aureus LuxS/AI-2 system for the first time, which raises the possibility that BAI could be employed as a possible antimicrobial drug to treat S. aureus strain-caused biofilms.

Assessment of the Physicochemical Properties of Fragrant Rapeseed Blended Hotpot Oil by Principal Component Analysis.

In this study, a nutritious, healthy Chongqing hotpot oil with excellent flavor was blended while considering nutrition, flavor, and health aspects. Four blended hotpot oils prepared from fragrant rapeseed, palm, sesame, and chicken oils were analyzed to determine their physicochemical properties, antioxidant capacities, levels of harmful substances, and nutritional compositions, and their sensory qualities were evaluated. Principal component analysis was performed to identify the best hotpot oil (10% chicken oil + 20% palm oil + 10% sesame oil + 60% fragrant rapeseed oil), which exhibited good antioxidant capacity (Oxidation Stability Index: 7.95 h; 2,2-diphenyl-1-picrylhydrazyl: 168.6 µmol/kg, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate): 116.7 µmol/kg, and ferric-reducing/antioxidant power: 63.9 µmol/kg), a high sensory score (7.7/10), stable physicochemical properties (acid value: 0.27 mg/g and peroxide value: 0.01 g/100 g), and high tocopherol (54.22%), and phytosterol retention (98.52%) after boiling for 8 h. Although the 3,4-benzopyrene content of this hotpot oil exceeded the EU standard after boiling for 7 h, the increase in the amount of harmful substances was the lowest.

Insights into the Effects and Mechanism of Andrographolide-Mediated Recovery of Susceptibility of Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

The frequent resistance associated with ß-lactam antibiotics and the high frequency of mutations in ß-lactamases constitute a major clinical challenge that can no longer be ignored. Andrographolide (AP), a natural active compound, has been shown to restore susceptibility to ß-lactam antibiotics. Fluorescence quenching and molecular simulation showed that AP quenched the intrinsic fluorescence of ß-lactamase BlaZ and stably bound to the residues in the catalytic cavity of BlaZ. Of note, AP was found to reduce the stability of the cell wall (CW) in methicillin-resistant Staphylococcus aureus (MRSA), and in combination with penicillin G (PEN), it significantly induced CW roughness and dispersion and even caused its disintegration, while the same concentration of PEN did not. In addition, transcriptome sequencing revealed that AP induced a significant stress response and increased peptidoglycan (PG) synthesis but disrupted its cross-linking, and it repressed the expression of critical genes such as mecA, blaZ, and sarA. We also validated these findings by quantitative reverse transcription-PCR (qRT-PCR). Association analysis using the GEO database showed that the alterations caused by AP were similar to those caused by mutations in the sarA gene. In summary, AP was able to restore the susceptibility of MRSA to ß-lactam antibiotics, mainly by inhibiting the ß-lactamase BlaZ, by downregulating the expression of critical resistance genes such as mecA and blaZ, and by disrupting CW homeostasis. In addition, restoration of susceptibility to antibiotics could be achieved by inhibiting the global regulator SarA, providing an effective solution to alleviate the problem of bacterial resistance. IMPORTANCE Increasingly, alternatives to antibiotics are being used to mitigate the rapid onset and development of bacterial resistance, and the combination of natural compounds with traditional antibiotics has become an effective therapeutic strategy. Therefore, we attempted to discover more mechanisms to restore susceptibility and effective dosing strategies. Andrographolide (AP), as a natural active ingredient, can mediate recovery of susceptibility of MRSA to ß-lactam antibiotics. AP bound stably to the ß-lactamase BlaZ and impaired its hydrolytic activity. Notably, AP was able to downregulate the expression of critical resistance genes such as mecA, blaZ, and sarA. Meanwhile, it disrupted the CW cross-linking and homeostasis, while the same concentration of penicillin could not. The multiple inhibitory effect of AP resensitizes intrinsically resistant bacteria to ß-lactam antibiotics, effectively prolonging the use cycle of these antibiotics and providing an effective solution to reduce the dosage of antibiotics and providing a theoretical reference for the prevention and control of MRSA.

Inhibitory Effect of Andrographis paniculata Lactone on Staphylococcus aureus α-Hemolysin.

We investigated the effect of andrographolide (AP) on the hemolytic capacity of Staphylococcus aureus (S. aureus) isolated from our region. AP is a labdane diterpenoid isolated from the stem and leaves of Andrographis paniculata. The hla gene from 234 S. aureus strains and the quality control standard strain ATCC29213 in dairy cows in some areas of Ningxia was analyzed. Evolutionary analysis, homology modeling, and functional enrichment annotation of α-hemolysin Hla detected from our region were performed through bioinformatics. The hemolytic ability of S. aureus isolates from the region was examined using the hemolysis test, and the effect of AP on S. aureus was quantified. Moreover, the effect of AP on the transcript levels of hla and genes highly related to hla (i.e., clfA and fnbA) was examined through fluorescence quantitative PCR. The mode of action of AP on the detected Hla was analyzed through molecular docking and dynamic simulation. The results showed that S. aureus in our region has a high rate of hla carriage. The hemolytic activity of strains NM98 and XF10 was significant, and ATCC29213 also exhibited some hemolytic activity. AP could inhibit the expression of Hla and its related proteins by downregulating hla, clfA, and fnbA transcript levels, which in turn attenuated the S. aureus hemolytic activity. Meanwhile, the AP molecule can form three hydrogen bonds with residues ASN105, SER106, and THR155 of Hla protein; bind with PRO103 through alkyl intermolecular forces; and form carbon hydrogen bonds with LYS154, reflecting that the AP molecule has a comparatively ideal theoretical binding activity with Hla protein. Among them, PRO103 and LYS154 are highly conserved in Hla protein molecules and play pivotal roles in the biological functions of Hla, and their binding may affect these functions. Their binding may also prevent the conformational transition of Hla from a monomer to an oligomer, thus inhibiting Hla hemolytic activity. This study offers a molecular basis for use of AP as an antivirulence drug and new ideas for developing novel drugs against S. aureus infection.

Potential Mechanisms of Quercetin Influence the ClfB Protein During Biofilm Formation of Staphylococcus aureus.

This study aimed to establish the mode of binding between Quercetin (QEN) and an essential protein called ClfB in forming biofilm in Staphylococcus aureus (S. aureus). In this study, the raw data of GSE163153 were analyzed for quality control, alignment, and gene counts, and the differential analysis detected the key differentially expressed genes (DEGs) assisting in the formation of the S. aureus biofilm. Then, the protein-protein interaction (PPI) and gene function enrichment analyses of the target genes, identified a gene called clfB to be closely related to biofilm formation. ClfB was structurally characterized, molecularly docked, and kinetically simulated to unravel the mode of binding of QEN to ClfB. Meanwhile, the growth curve and transmission electron microscopy methods examined the effect of QEN on the S. aureus growth. Results indicated that the clfB gene was increasingly expressed during biofilm formation and was involved in cell adhesion, pathogenicity, and infection. We identified 5 amino acid sites of ClfB (D272, R331, I379, K391, E490) as potential sites for binding QEN, which would indirectly influence the changes in the functional sites N234, D270, Y273, F328, inhibiting the formation of biofilm. Meanwhile, 128 µg/ml of QEN could significantly inhibit the S. aureus biofilm formation. This manuscript serves as a molecular foundation for QEN as an antibacterial drug providing a new perspective for developing antibacterial drugs.