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Objective:To investigate the inhibitory effects and mechanism of Reyanning mixture （RYN） combined with linezolid （LNZ） against methicillin-resistant <italic>Staphylococcus aureus</italic> （MRSA） and its biofilm. Method:The minimum inhibitory concentrations （MICs） of RYN and LNZ against MRSA were determined by microdilution assay. The microplate method was used to detect the changes in viable count before and after MRSA administration at four time points （0， 6， 12， 24 h） in the process of biofilm growth. The morphological changes of MRSA after 24 h were observed by scanning electron microscope. Metabonomic technique was applied to analyze the changes in terminal metabolites of endogenous small molecules from MRSA treated by the two drugs at four time points. Result:The MICs of RYN and LNZ were 1/2 of the stock solution concentration and 4 mg·L<sup>-1</sup>， respectively. The inhibitory effect of LNZ （2 mg·L<sup>-1</sup>） against viable bacteria at 0 h was better than that of 1/16 RYN. At 6， 12， 24 h， 1/16 RYN was superior to LNZ in inhibiting MRSA. The inhibitory effects of RYN combined with LNZ were better than those of RYN or LNZ alone at the four time points. RYN combined with LNZ caused more severe damages to the morphological structure of MRSA biofilm at 24 h than RYN or LNZ alone. Cyclic adenosine monophosphate （cAMP）， adenosine diphosphate （ADP）-<italic>D</italic>-ribose and 2-methylbutanoyl-coenzyme A （2M-CoA）， as the metabolites related to biofilm formation， were immune to LNZ， but 2M-CoA and ADP-<italic>D</italic>-ribose were influenced by RYN at 12 h and 24 h. The combined use of RYN and LNZ interfered with the three metabolites at 24 h. <italic>L</italic>-tryptophan， phenylpyruvic acid， cytidine and sebacic acid were the pharmacometabolic markers of LNZ， and the related biological pathways were phenylalanine， tyrosine and tryptophan biosynthesis and phenylalanine metabolism. Four metabolites such as<italic> L</italic>-histidine， uric acid， and <italic>L</italic>-lysine were the pharmacometabolic markers of RYN， with phenylalanine metabolism and aminoacyl-transfer ribonucleic acid （tRNA） biosynthesis confirmed as the related biological pathways. Nine metabolites such as <italic>L</italic>-tryptophan，<italic> L</italic>-lysine， and sphingosine-1-phosphate were responsible for the efficacy of RYN combined with LNZ. The related biological pathways involved aminoacyl-tRNA biosynthesis， phenylalanine， tyrosine and tryptophan biosynthesis， novobiocin biosynthesis， and tyrosine metabolism. Conclusion:RYN combined with LNZ better exerts the inhibitory effects against MRSA at each time point of its biofilm formation， which is attributed to cAMP metabolism. The synergistic effect resulted from aminoacyl-tRNA biosynthesis and phenylalanine， tyrosine and tryptophan biosynthesis. RYN combined with LNZ can serve as a potentially effective solution to MRSA infection.
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Objective To further explore the genetic characteristics of oidiomycetes mutant strains like bacterial morphology on the basis of the study on morphology and structure of mutated candida.Methods The standard strains of candida albicans were induced by low temperature and under the condition of low temperature and nutrient deficiency.Variation of standard strains of Candida albicans were induced by clinical antifungal drugs such as fluconazole with different concentration gradient.Fungal gene template was prepared by boiling method,sequences of 16SRNA and 18SRNA were amplified using bacteria conservative gene sequence of 16SRNA and fungal conserved gene sequence of 18SRNA,and observed and recorded the results agarose gel electrophoresis.At the same time,the amplified fragment of bacterial conservative gene 16SRNA was sequenced,and the sequence was analyzed by BLAST comparison.Results the 16SRNA sequences of candida variant were amplified positive,while the standard strain of candida albicans did not show the corresponding amplification band.Except 2 strains which showed a faint band,the other variants of the 18SRNA sequences did not amplified the target band,while the standard strains of candida albicans showed a corresponding amplification bands.Suggested that proportion of 18SRNA sequences in the genome of oidiomycetes mutant strains like bacterial morphology was not much even lack.The 16SRNA fragments amplified of oidiomycetes mutant strains like bacterial morphology did determination of DNA sequence after purification.BLAST comparison analysis,it was found that sequence of oidiomycetes mutant strains like bacterial morphology had higher similarity with bacterial sequences in the database.Conclusion Oidiomycetes mutant strains like bacterial morphology contained bacterial and a small amount of fungus conservative gene.Oidiomycetes mutant strains like bacterial morphology with original nuclear biological character are ones from eukaryotes.This study is great significance in biological evolution,especially in the evolution of prokaryotic cells and eukaryotic cells.
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Objective To investigate the changes of the morphology,structure and biological characters of mutated Candida and through its genetic characteristics,research and reveal the mechanism of the variation at the molecular level.Methods Used different nutritional conditions,different growth conditions and different azole antifungal agents to induce mutation of the standard strains of Candida albicans.In clinical study,Candida mutant strains was isolated from vaginal secretions,pleu-ral effusion and gastric juice samples in patients of poor effect with Antifungal therapy,and studied on the morphological characteristics,and the nuclear structure,the biochemical reaction,the drug resistance,the bacterial composition and the ge-netic characteristics of above variants,etc.Results Mycelial?morphology:Candida were prone to mutation like bacteria, mutant bacteria could show G+ Aureus shape,G+ Bacillus,G+ long filamentous,G- Aureus shape,G- Bacillus and G- long filamentous;Nuclear structure:Candida mutant strains changed like prokaryotes under the electron microscope because it lost the original structure of eukaryotic cells.Biochemical reaction:there were 5 different items in 20 biochemical test ob-served.Drug sensitivity test:Candida mutated to antifungal drugs being originally sensitive was completely resistant,sensi-tive and resistant originally was completely sensitive,and the same as ordinary bacteria resistant.The cell component chan-ges:there was significantly different in Candida variant strain and the atavism of variant strain identified by mass spectrome-try.The most conservative fungalgene expression:Candida mutated had conservative gene expression of eukaryotes.It could be demonstrated that oidiomycetes mutant strains like bacterial morphology with prokaryotic cell biological characteristics was derived from Candida with eukaryotic cells.Conclusion Candida was prone to variation like bacterial morphology.The biological characteristics of mutant resembled prokaryote.There was a qualitative change among the standard strains of Can-dida albicans,mutant strains of oidiomycetes like bacterial morphology and the atavism of variant strain with clear genetic re-lationship under the electron microscope in the form of nuclear matter.The study on biological evolution,especially contact in prokaryotic cells and eukaryotic evolution has very important significance.
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Objective To evaluate the antibacterial activity in vitro of Galla Chinensis ethanol extractant(GCEE)against S.epidermidis,and the change of cell morphology of S.epidermidis.Methods Deterimination of antibacterial activity of GCEE against 124 strains S.epidermidis was performed by using agar dilution method,and the changes of cell merphalogy of S.epidermidis was examined by microscope.Results The MIC90 of GCEE against 43 strains of MRSE(methicillin-resistant staphylococcus epidermidis)and 81 strains of MSSE(methicillin-sensitive staphylococcus epidermidis)were 0.288 and 0.144 mg/mL respectively.Under microscope,the cells of S.epidermidis were expansion by degrees,with the edge of cell of S.epidermidis irregular.The substances in cells of S.epidermidis were disappear final.Conclusion Galla Chinensis ethanol extractant had strong antibacterial activity against 124 strains of S.epidermidis,and the cell morphology of S.epidermidis changed.