| PrefaceKlebsiella pneumoniae is responsible for opportunistic infections, particularly of the respiratory tract and intestinal tract, in humans. It can adhere to interface and secrete glycocalyx to form biofilm. Biofilm acts as a barrier to protect the infecting cells from host defense system and antibiotics, which makes some infections intractable and relapsing. Klebsiella pneumoniae biofilm is very common in chronic airway infections, and the infection of biofilm has attracted the attention of clinical doctors.We must establish an ideal model to study the biofilm bacteria. Compared with models in vitro, an in vivo model is beneficial to consider the influence of internal environment. We are to establish the Klebsiella biofilm model of pulmonary infection in guinea pigs in an inhalation method, then we will investigate the evolution of Klebsiella pneumoniae biofilm in vivo by quantifying bacteria and observing through light and electron microscopy firstly. Secondly, we are to e-valuate the efficacies of cefotaxime( CTX) and the combination of CTX and roxithromycin( RXM) , which would be beneficial to the treatmentof Klebsiella pneumoniae biofilm - associated infections.Methods1. The animals were divided into four groups. 10 animals in group A were inoculated with saline, 30 animals in group B were inoculated with the suspension of Klebsiella pneumoniae, 5 animals in group C were treated with CTX and 5 animals in group D were treated with the combination of CTX and RXM after inoculation by Klebsiella pneumoniae.2. The guinea pigs were subcutaneously treated with dexamethson at a dose of 3mg/kg/day for seven consecutive days before inoculation. Five animals in each group were placed in a plastic chamber e-quipped with a nebulizer. All animals were inoculated by physiological saline solution or the bacterial suspension of which the concentration was adjusted to 6. 25 x 108 colony forming unit( CFU/ml) . Eight mil-liliters of bacterial suspension was dispersed through the nebulizer for 40 minutes at an atomospheric pressure of about 0.765kg/cm2.3. On day 7 after inoculation, animals in group C were subcutaneously treated with CTX at a dose of 50mg/kg twice a day for 3 consecutive days. Animals in group D were treated with oral administration of RXM at a dose of 2.5mg/kg twice a day and subcutaneous injection of CTX at the same dose.4. All animals were sacrificed by cardiac puncture on the sampled days. The lung tissues were aseptically removed and weighted, then homogenized with 1ml of saline. Serial tenfold dilutions were made with saline, and 10ul of each dilution was cultured on blood agar plate overnight at 37 C. Colonies grown on blood agar plate wereidentified routinely and counted, the colony forming unit (CFU)/g -lung was quantified.5. After sampled, lung tissues were fixed with 10% formalin and 2.5% glutaraldehyde respectively, then observed with light microscopy and scanning electron microscopy ( SEM) .6. We used analysis of variance as the statistic method to deal with these data in group B, C, D on day 10.Result1. Almost all the animals survived to the sampled days after inoculation except for two died animals in group B on day 1.2. No organisms were detected from the lung tissues in group A during the whole experiment. The organisms in group B after the 3rd day were smaller in colony, slower in growth, shorter in length than those before inoculation. Under light microscopy, Klebsiella pneu-moniae biofilm existed in the form of tubercles which consisted of poly-morphonuclear cells (PMNs) , lymphatic cells, mononuclear cells and so on. The outer layer of granulation tissues consisted of fibroblasts and epithelioid cells. Under SEM, on day 7 after inoculation, the bacteria were seen enclosed by glycocalix in the tubercles and aggregated to each other through fibrous structure, and lymphatic cells could be seen in the biofilm. on day 10, bacteria clustered , and glycocalix was more than that on day 7.3. On day 10 after... |
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