The Influence Of Anti-alginate Serum On Biofilm Of Mucoid Pseudomonas Aeruginosa

OBJECTIVE The influence of anti-alginate serum combined with gatifloxacin on biofilm of mucoid Pseudomonas aeruginosa was investigated. METHODS The anti-alginate serum was prepared in rabbits immuned with alginate-bovine serum albumin and potency activity was determined by ELLSA method; The modified plate culture method was used to establish biofilm model in vitro and the biofilm was treated by series of dilutions of anti-algiante serum or combined with different concentrations of gatifloxacin, The samples were then detected by scanning electronic microscopy (SEM) and the optical microscopy after being stained by AgNO3, the optical density of bacterial biofllm was determined by using computer image analysis system; The adherence of mucoid Pseudomonas aeruginosa was determined by crystal violet stained method; the gatifloxacin penetration activity was determined by concentration gradient penetration method; The viable counts of bacteria in BF were detected by MTT method; . The minimal biofilm eradication concentration (MBEC) was determined by micro-dilution method. RESULTS The BF on silicon slides was achieved after incubation of mucoid Pseudomonas aeruginosa in trypcase soy broth (TSB) after 7 days. Microscope graphs of mucoid Pseudomonas aeruginosa biofilm showed that cluster bacilli surrounded by dense black stained material, The SEM graphs showed that bacteria were bacilus surrounded by accidented material and the bacteria were connected each other by shines to form biofilm .Higher titer of anti-alginate serum(titer 1:320) was obtained from rabbits immuned by alginate-BSA. The computer image analysis system showed that the mean optical density of bacterial biofilm was reduced from 0.82 ?0.06 to 0.70 ?0.04 when 1 x MIC gatifloxacin combined with 1:1 dilution anti-alginate serum. The graphs of microscope and SEM showed slime become sparse and the shape of bacteria become irregular. When 1:1 dilution anti-alginate serum combined with 1 xMIC~ 4xMIC and 8xMIC gatifloxacin , the adherence of mucoid Pseudomonas aeruginosa decreased significantly , the optical density of silicon slides at 540nm decreased from 0.130?.010,0.129?.015 and 0.126?.011 to 0.120? 0.010,0.117?.015 and 0.1140?.010. The penetrated concentrations of gatifloxacin through biofilm were increased from 1.210 ?0.091,2.911 ? 0.112 and 5.911 ?0.213 to 1.752 ?0.122,3.908 ?0.154 and 7.898 ?0.321 when 1:1 dilution anti-alginate serum combined with 45.1 gIml ,8 ~ glml and 16 ~t gIml gatifloxacin . 1:1 dilution anti-alginate serum combined with 1 xMIC,4xMIC and 8xMIC gatifloxaeinalso decreased viable counts in biofilms significantly from 6.63 ?0.85~ 6.48 ?1.12 and 5.73 ?1.10 lgcfulcm2 to 5.76 ?0.90,5.02 ?0.93 and 4.70 ?0.62 lgcfulcm2. 1:1 dilution anti-alginate serum decreased the MBEC from 8 5.1 gIml to 4 ~i gIml of gatifloxacin. CONCLUSION The anti-alginate serum could affect the structure and formation of the biofilm of mucoid Pseudomonas aeruginosa ,decrease the adherence of Pseudomonas aeruginosa to silicon slides , enhance the 12 penetration of gatifloxacin to Pseudomonas aeruginosa through biofilm decrease the MBEC of gatifloxacin to Pseudomonas aeruginosa in biofilm. The results suggested that synergistic effect was observed in anti-alginate serum combined with gatifloxacin.