| ObjectiveA strain of Staphylococcus aureus was isolated from a clinical case and had strong host invasiveness.In order to explore the mechanism of enhanced host invasion ability WLR Staphylococcus aureus new strain,we studied the effect of lipase activity onbiofilm formation and host invasion ability.Methods1.The bacteria WLR bacteria were isolated and cultured from the skin wound of the patients by aseptic operation,and their biological characteristics were analyzed by plasma coagulase experiment,glycolysis experiment and drug sensitivity test.Identification of bacterial species by 16 S r DNA of gene sequence sequencing and genome sequencing.2.The animal models of subcutaneous and abdominal infection were established to detect the signs and distribution and reproduction of visceral organs in infected mice.3.Cell number of new strain WLR and standard strain was detected by oleic acid sterilization experiment.4.After 24 hours of culture in liquid medium containing or without 100μmol/L glycerol trioleate,or 1μg/L farnesol,biofilm were quantified by crystal violet staining.5.The 10~8bacteria were treated with 100μm glycerol trioleate or 1μg/L farnesol for24 h and interact with 10~6A549 cells for 1.5 h,and then the cells treated with 1.5 h lysozyme.Next,the cell Lysis solution were cultured in LB agar after 10~4dilution.Results1、The pus line facultative anaerobic culture 24 h,formed moist,smooth surface,neat edge,showing white central protruding diameter of about 1.0 mm round colony on the LB medium,bacteria into grape string arrangement,Gram-positive cocci.The plasma coagulase test was positive.The results sr DNA sequencing Blast comparison showed that Staphylococcus aureus was named WLR strain.WLR maltose can becatabolized,mannitol fermentation is negative,other monosaccharides,sucrose,lactose and glucose can be fermented and metabolized.the sensitivity of WLR to compound sinomine,norfloxacin,gentamicin,erythromycin,penicillin G was significantly reduced compared with that of ATCC25923 bacteria,and the sensitivity of streptomycin was not significantly reduced.2、WLR strain can cause 0.8 X1cm size of skin damage infection characteristics consistent with the patient’s skin lesions,and Staphylococcus aureus standard strain ATCC25923 caused by 0.3 X0.5,significantly less than the WLR strain,indicating that the strain is more invasive than 25923 strain.3、The liver,kidney and spleen of mice infected with WLR strains were all affected,ranging from 105 to 107 CFU and only one-third of the mice inoculated with ATCC25923 form abscesses.4、The total DNA length of WLR strain was 2744392 bp,and the number of predicted genes was 2608.17 gene mutations were related to virulence enhancement,including lipase gene.5、Bacteriostasis test of oleic acid showed that compared with 25923 strain,WLR bacteria significantly resisted the bactericidal effect of oleic acid.At the same time,we found that there were WLR bacteria that were significantly resistant to oleic acid,and their lipase activity was also significantly increased.6、Compared the activity of bacterial lipase in strains WLR and 25923 after 100μm Tri O treatment for 24 h,and found that the activity of lipase in strains of 25923 and untreated group was significantly higher(P<0.01).Conversely,the lipase activity of the WLR and 25923 strains was significantly lower than in the untreated group after farnesol treatment(P<0.01).7、Tri O pretreatment activated lipase activity and allowed bacteria to adhere and invade A549 cells.the results showed that the invasion ability of WLR strains after increased lipase activity was significantly enhanced than that of 25923 strain and untreated group(P<0.01),respectively.In contrast,the invasion ability of WLR strains were significantly lower than untreated groups after farnesol treatment(P<0.01).Conclusions1.A new isolated Staphylococcus aureus WLR has stronger invasive abilitythan standard strain 25923.2.Lipase activity is associated with biofilm formation and host cell invasion WLR Staphylococcus aureus. |
