| Sta. aureus is a major pathogens of mastitis, exists mainly in the form of biofilm in the mammary gland. Studying the biofilm formation ability of Sta. aureus and related factors is significant for screening clinical drug and the epitope vaccine for biofilm inhibition. In this study, Sta. aureus biofilm research results are as follows:1. Biofilm formation and analysis of associated genes involved in Staphylococcus isolates from bovine mastitisStaphylococcus biofilms formation were conducted using a silicone elastomer slices plate assay in 24-well plates, after rinsing planktonic bacteria away, the biofilm forming abilities of them were determined by silver staining qualitatively and by crystal violet staining quantitatively, and the structure of biofilm was observed through scanning electron microscopy. Formation of biofilm could be found macroscopically in 120 out of 137 strains by silver staining, and the biofilm formation rate was 87.6%. It showed that 5 strains didn't adhere to the surface of silica gel by crystal violet staining, while the rest 132 isolates did. bap, icaAD, icaBC, sar, agr, sigB, clfaA, clfaB, fnbpA and fnbpB were amplified by PCR. bap was amplified in 57 isolates and icaAD and icaBC in 43 and 54 strains respectively; sigB, sar and agr were amplified in 73, 49 and 38 isolates, and clfaA and clfaB in 76 and 50 strains respectively; fnbpA was present in 52 strains and fnbpB in 26 isolates. It reveals that bap, sigB, sar, icaAD and icaBC may be significant biofilm-associated genes, for these genes are present more in biofilm-positive strains than in biofilm-negative strains. The roles of agr,clfaA,clfaB,fnbpA and fnbpB genes in biofilm development are unassured.2. The effect of antibiotics to biofilmsBiofilms formation of e9 and e25 strains were conducted using a silicone elastomer slices plate assay in 24-well plates, one milliliter of sterile TSB contained antibiotics at 1/16 of minimum inhibitory concentration and ten microliters of overnight cultured Staphylococcus strain were added to each well. Incubated at 37℃in biochemical incubator for 3 days and changed the medium every 12 hours. After incubation, biofilms were strained with 200μl 2% crystal violet and measured by reading the optical density at 492nm. Cefepime significantly inhibited the biofilm formation of strong-biofilmed strain e9, while Ampicilin and Methicilin enhanced the formation of biofilm instead of inhibition; when it came to weak-biofilmed strain e25, two kinds of antibiotics hardly inhibite the biofilm formation, Methincilin did obviously.Put three-day and seven-day biofilm of experimental strains into the TSB with various antibiotics in it, incubated at 37℃for 1 day and change the medium per 12 hours with fresh TSB containing antibiotics. For the strong-biofilmed strain e9, in addition to a low concentrations (1/16MIC) of Cefepime, other concentrations of antibiotics all inhibited its three-day biofilm, and the effects of Ampicilin and Cefepime at ten times of minimum inhibitory concentration were very obvious(P<0.05); All other antibiotics inhibited seven-day mature biofilm expect Ampicilin at minimum inhibitory concentration, and the higher the concentration were, the inhibitory effect of antibiotics were better. When it came to weak-biofilmed strain e25, Amp at 1/16 of minimum inhibitory concentration almost didn't have inhibition for three-day biofilm, while other antibiotics did, but not significantly; The seven-day biofilm had a similar situation, the effect of antibiotics at minimum inhibitory concentration was weak, and that of higher concentration antibiotics were obvious. All three concentrations of Methicilin and Cefepime were able to inhibite seven-day biofilm, and the groups treated with Methicilin and Cefepime at twenty times of minimum inhibitory concentration were extremely significantly different form the control groups(P<0.01). The results showed that higher concentrations antibiotics performed better on inhibition biofilm formation than those at lower concertrations, and immature biofilms were more easily to inhibite. The presence of low concentration of Amp and Met almost had no inhibitory effect, even stimulated or promoted biofilm formation, and the stronger biofilm formation capacity of strains were, the promotion were the more apparent.3. Construction of T7 phage display cDNA library from Holstein cows mammarymRNA was extracted and isolated using Magnetight Oligo(dT) Particles in Straight A'sTM mRNA Isolation System, and then, the agarose gel electrophoresis showed the range of mRNA size. After mRNA was reverse-transcribed into cDNA,end modification,Directional EcoR I/Hind III Linkers ligation and Hind III and EcoR I digestion were performed. The double-strand cDNA fragments longer than 300bp in length were fractionated by the Mini Column, and ligated into the T7Select10-3b vector with EcoR I and Hind III adhering ends. After packaging in vitro, the recombinant T7Select10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. There were 6×10~5 recombinants in the initial cDNA library.
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