| Staphylococcus aureus(S.aureus)is a Gram-positive pathogen that poses a serious threat to livestock and human health.Currently,the emergence of antibiotic-resistant S.aureus,especially methicillin-resistant S.aureus(MRSA),has complicated the treatment of S.aureus infections,and therefore there is an urgent need for the development of new drugs and innovative strategies to combat S.aureus infections.However,the quorum-sensing(QS)system plays a key role in the regulation of bacterial drug resistance,and therefore,new antimicrobial strategies based on the inhibition of bacterial QS have emerged as a promising approach for attenuating bacterial pathogenicity and preventing bacterial resistance to antibiotics.In recent years,lactic acid bacteria(LAB)have been shown to produce a variety of antimicrobial substances and exist in their cell-free supernatant(CFS),enabling LAB-CFS to exhibit favorable antibacterial activity.At the same time,many traditional Chinese medicines and their active ingredients play anti-inflammatory and immunomodulatory roles in many diseases,and are widely used in disease control in livestock and poultry farming,and are the first choice of livestock and poultry feed additives after the ban of antibiotics,and the discovery of lead compounds for the research and development of new drugs for "replacement of antibiotics".It is the first choice of livestock and poultry feed additives after the ban of antibiotics and an important source for the discovery of lead compounds for new drug development Therefore,this study focused on LAB-CFS and baicalin(BAI),the active ingredient of traditional Chinese medicine Scutellaria baicalensis,to explore the effect of the LuxS/AI-2 QS system on the control effect of S.aureus infection when them act in combination.1.The production of AI-2 in S.aureus was examined by optimizing the conditions for the detection of AI-2 signaling molecules,screening for strains with high production of AI-2,and evaluating the effects of pH,temperature,NaCl,and nutrient-induced stress conditions on the growth and AI-2 activity of the S.aureus Wld10 strain.In addition,RT-qPCR was used to compare the transcript levels of luxS and pfs genes after different environmental stresses.The results showed that AI-2 was best detected with freshly cultured V.harveyi BB170 when co-incubated for 5 h with CFS prepared from logarithmic growth stage S.aureus cultures.Moreover,the ability of strain Wld10 to produce AI-2 as well as the luxS gene expression level were significantly better than the other strains.Under the stress environment,the bacterial growth was vigorous under alkaline condition and induced the production of AI-2 in S.aureus;under osmotic pressure and low temperature stress,the bacterial density and the production of AI-2 in S.aureus were inhibited at the same time,but the low nutrient stress could induce the production of AI-2 in S.aureus.The expression of luxS and pfs genes of S.aureus changed to different degrees in different environmental stresses.S.aureus under different environmental stresses had different degrees of changes in luxS and pfs gene expression.2.In order to investigate the effect of CFS prepared from LAB in planktonic and biofilm states on the activity of S.aureus,their effects on growth,biofilm,virulence factors and antibiotic sensitivity of S.aureus were evaluated.The results revealed that LAB-CFS treatment significantly inhibited the growth,biofilm,and cellular physiological properties of S.aureus(p<0.05),including hydrophobicity,extracellular polysaccharide(PIA),and eDNA production,and down-regulated the expression levels of genes encoding biofilm and virulence factor-related genes,and also increased the S.aureus sensitivity to antibiotics.Moreover,the effect of CFS prepared by LAB in the biofilm state was superior to that of CFS prepared by LAB in the planktonic state.3.To preliminarily investigate the effect of BAI on the LuxS/AI-2 QS system to regulate S.aureus infection,firstly,we used molecular docking technique to explore the binding activity of BAI with LuxS protein and examined the conformation of LuxS protein after BAI treatment,and then we explored the MIC and growth curves of the strains after the action of different concentrations of BAI through detailed in vitro experiments,and we used microscopy to the inhibitory effect of BAI on S.aureus biofilm was visualized and its removal effect on S.aureus mature biofilm was detected.Meanwhile,RT-qPCR method was used to analyze the effects of BAI on the expression of S.aureus biofilm and virulence-related genes,and to explore the changes of hemolysin activity,AI-2 activity,and susceptibility to antibiotics in S.aureus during the action of BAI.The molecular docking results showed that BAI formed a stabilizing complex with amino acid residues in the active site of LuxS protein through the formation of hydrogen bonds,pi-Alkyl and pi-pi T-shaped interactions.Meanwhile,BAI had a weak inhibitory effect on S.aureus in the planktonic state with a MIC of 1024 μg/mL,but significantly reduced the formation of S.aureus biofilm and promoted the dispersion of mature biofilm,and at the same time,it was able to increase the susceptibility of S.aureus to Penicillin,Ciprofloxacin and Gentamicin,decrease its hemolytic activity,downregulate S.aureus biofilm,and concentration-dependently down-regulated the expression levels of biofilm and virulence-related genes and the activity of AI-2 in S.aureus.4.The recombinant proteins LuxS and Pfs were constructed to synthesize AI-2 in vitro,and the conditions of AI-2 in vitro synthesis were optimized,meanwhile,the effect of adding exogenous AI-2 on S.aureus was detected.The results revealed that soluble recombinant proteins LuxS and Pfs were successfully obtained,and when the purified recombinant proteins were co-incubated with SAH,the products were able to induce luminescence of V harveyi BB170,suggesting that biologically active AI-2 was synthesized.Addition of exogenous AI-2 was able to stimulate the growth of S.aureus colonies,promote the formation of the S.aureus biofilm and increase the production of virulence factors.5.The antibacterial activity of LAB-CFS with BAI alone and in combination was determined by checkerboard assay and time-kill curve.The anti-biofilm activity of LAB-CFS with BAI alone and in combination was observed by scanning electron microscopy.Meanwhile,a model was established to evaluate the effect of LAB-CFS and BAI on mastitis in mice by inducing mastitis using milk duct injection of S.aureus.The results showed that the combination of LAB-CFS and BAI had better inhibitory activity against S.aureus than alone.After treatment with both LAB-CFS and BAI,the density of S.aureus biofilm showed significant reduction,and the combined inhibition of S.aureus biofilm was the most effective.Meanwhile,the combination significantly reduced the inflammatory damage of breast tissue,inhibited myeloperoxidase activity and the secretion of inflammatory factors IL-1β,IL-6,and TNF-α and the expression of TLR2 and NF-p65 was also significantly inhibited.6.Liquid chromatography-mass spectrometry(LC-MS)was used to analysis of the effects of adding LAB-CFS with BAI on the metabolome of S.aureus.It was found that peptidoglycan and phosphomimetic acid metabolic pathways were significantly altered in S.aureus after treatment,while TCA cycle-related metabolism,amino acid and nucleotide metabolism were also altered,and that the combination of LAB-CFS and BAI reflected a significant disruption of bacterial membrane and cell wall biosynthesis.This study reveals that LAB-CFS and BAI,when used in combination,can produce a synergistic effect with favorable in vitro bacterial inhibitory properties and significant in vivo antimicrobial activity,and also suggests that the combination influences the regulatory effects of the LuxS/AI-2 QS system on S.aureus infection.These findings provide insights into the potential mode of action of LAB-CFS and BAI and deepen our understanding of the regulatory function of the LuxS/AI-2 QS system.Thus,LAB-CFS and BAI could be potential alternatives or adjuvants to conventional antibiotics. |
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