| It is well known that chronic airway infection caused by Pseudomonas aeruginosa is one of the most difficult infections to treat. When sensitive anti ?Pseudomonas agents are used, bacterial eradication is hardly obtained, even if transient improvement of clinical symptoms is observed. The patients repeatedly suffer from symptom recurrence. The existence of biofilm - covered bacteria plays an important role in the intractable infection. Biofilm is the matrix - enclosed bacterial populations adherent to each other and/or to surface or interfaces. Biofilm may shield the bacteria from the killing of antibiotics as well as the immune system of the hosts. Up to now, domestic research of biofilm is mainly focused on in vitro studies, there is still no report on the in vivo model of biofilm in China. Our study aimed to establish an in vivo model of Pseudomonas aeruginosa biofilm in lung, with which the histopathological and bacteriological features of this kind of infection and the effects of antibiotics could be investigated.Methods1. Establish the model of Pseudomonas aeruginosa biofilm inlung: A clinical isolate of Pseudomonas aeruginosa was incubated overnigt at 371 , then the bacteria were suspended in saline and the bacterial concentration was adjusted to 6. 25 x 108CFU/ml. Guinea pigs were subcutaneously treated with dexamethasone at a dose of 3mg/kg/day for 7 consecutive days before inoculation. The animals were placed in a plastic chamber equipped with a nebulizer on the side and the bacterial suspension was dispersed through the nebulizer for 40 minutes. Thus, Pseudomonas aeruginosa were inspired into the lung of the guinea pigs in the form of aerosol. Animals in control group ( group A) were challenged with sterilized normal saline. 3 ,7,10,14,17and 21 days after challenge, 5 animals were sacrificed on each day and the lungs were removed aseptically.2. Estimate the colony forming unit(CFU)in lung: Samples of lung tissue of one side were lavaged with sterilized saline though bron-chi , then the samples were homogenized with 1 ml of sterilized saline. Then, tenfold dilutions were made, and lμl of each dilution was cul-tured on blood agar plates overnight at 37?C. The colonies were coun-ted and identified on the next day.3. Observe the specimen of lung under microscope. Immediately after the animal were sacrificed, part of the lung tissue were sampled, fixed with 10% buffered formalin and stained with hematoxylin and eo-sin( HE) , then observed under light microscope. Another part of lung tissue were fixed with 2. 5% glutaraldehyde, after being splitted in liquid nitrogen and undergoing a series of procedures, the specimens were observed under scanning electron microscope (SEM).4. Detect the efficacy of antibiotics. 15 animals were divided into 3 groups which are the group challenged with Pseudomonas aeruginosa but not treated with any antibiotics ( group B) , the group treated withCTX( group C) and the group treated with both CTX and RXM( group D). CTX was given subcutaneously( l00mg/kg/day) twice a day and RXM was administrated(5mg/kg/day) twice a day orally.5. Statistical analysis, analysis of variance (SNK test) was per-formed.Results1. Bacteria were detected from all the animals challenged with Pseudomonas aeruginosa on each sacrifice day and the cultured organ-isms were identified to be Pseudomonas aeruginosa. While in the con-trol group, there was no bacterium detected.2. Under light microscope, scattered tubercles were seen in the lung tissues of infected animals, and the tubercles consisted mainly of lymphocytes, surrounded by flat epithelioid cells and fibroblasts, forming a kind of chronic granulomatous lesion.3. Under SEM, a great number of bacteria were seen in the tu-bercles, and the bacteria aggregated to each other were enveloped in mucoid matrix where lymphocyte scattered. With the elongation of time, the matrix of biofilm became thickened.4. The number of viable bacteria in the lung tissues of group C... |
PDF Full Text Request