Formation Characteristics And Drug Clearance Of Bacterial Biofilms In Suppurative Otitis Media Rats

PART Ⅰ Formation Characteristics and Significance of Bacteria Biofilm in Middle Ear Mucosa of Rats with Suppurative OtitisObjective:To establish the rat model of suppurative otitis media and observe the formation of bacteria biofilm in middle ear mucosa of animal models and discuss the role of bacteria biofilm in the pathogenesis of chronic suppurative otitis mediaMethod:28rats were divided into6experimental groups and a control group averagely. All rats in experimental groups were infected with pseudomonas aeruginosa solution in1×106CFU/lm concentration through the tympanic membrane puncture approach to bilateral middle ear cavity. On the1st,6th,10th,15th and21st day after inoculation respectively,4rats in one experimental group were narcotized, then two-sided tympanic membrane of each rat were observed by using electric otoscope. We rated on the severity of the inflammation from the general pathology level(0for normal,4for the most serious).After the execution, took the two-sided otocyst, the left was made to SEM specimen and was observed the shape of bacterial biofilm in middle ear mucous.The right was observed by CLSM. Control group were executed at the beginning of the experiment. Result:(1) Bacteria biofilm in line with their respective criteria were fond at the6th day after the inoculation, and were more typical in shape at the10th day. Then the states were maintained stably in3weeks.(2) Using electric otoscope to observe tympanic membrane, it can be seen that the inflammation severity of otitis media aggravates gradually in the first10days and achieves the peak, then the state will continue to the3rd week. The differences of tympanic membrane rating between1day group and6day group,6day group and10day group were statistically significant.(t=2.55,t=3.99; P<0.05,P<0.01)Conclusion:(1) In this experimental, the forming process of bacteria biofilm in rats model is:from beginning to the5th day, the bacteria adheres and accumulates. At the6th day, the3D structure of bacteria biofilm preliminary formatted. At about the10th day, the bacteria biofilm achieves the mature and steady state.(2) With the growth and maturity of bacterial biofilm in middle ear mucosa, the inflammation of otitis media is gradually increasing, which suggests that the inflammation severity of otitis media and the maturation of bacterial biofilm in middle ear mucosa are closely related. PARTⅡThe Eliminating Effects of Clarithromycin and Ciprofloxacin in Combination on P.aeruginosa Biofilms of Rats Middle Ear MucosaObjective:Using ciprofloxacin (CPFX) and clarithromycin (CAM) locally in rat tympanum after Pseudomonas aeruginosa (PA) biofilm formed to observe the effect of fluoroquinolone itself and the combination of fluoroquinolone and macrolides in eliminating PA biofilm.Method:36rats were randomly divided into3groups:The control group (A), CPFX group (B), CPFX+CAM combination group (C group), n=12. All rats were injected PA bacterial medium into the right tympanic cavities by tympanic puncture to cause suppurative otitis media models.10days later, the three groups were injected saline, CPFX liquid and CPFX-CAM mixed liquid into the right tympanic cavities respectively once a day. At the day1st,3rd,8th,14th after first administration of drugs,3rats of each group were sacrificed and tympanic bullas were taken out. After a few steps of treatment, the middle ear mucosa specimens were observed by a scanning electron microscope. The SEM images were analyzed by the software ImageJ and then the coverage rates of the bacterial biofilms on mucosal surface were obtained.Result:(1) The coverage rates of the bacterial biofilms of group A and B were maintained at about90%on the day3rd,8th,14th in the experimental section and the differences had no statistical significance at the three time points. There also was no statistical significance at the same time points between the two groups.(2) In group C, the coverage rates at the three time points were88.11±6.09%,33.94±13.53%and19.52±9.34%respectively, which has a decreasing trend. Three pairwise comparisons, the differences were statistically significant (tc3&c8,tc3&c14,tc8&c14were respectively14.14,23.83,3.40, all P<0.01,).(3) On the day3rd, there was no statistical significance in the coverage rate compared group C with group A and B. On the day8th and14th, group C had a smaller coverage rate than group A and B at the same time points, and the differences between A and B, A and C were statistically significant (tc8&A8, tc8&B8, tc14&A14, tc14&B14were respectively15.36,15.03,27.26,27.97, all P0.001)Conclusion:After the PA-induced biofilm formation in rat middle ear mucosa, the combination of CPFX and CAM was significantly effective in eliminating biofilm after being used for more than8days, while CPFX itself was not that much effective.