The purpose of this study was to develop a biofilm model of Klebsiella pneumoniae B5055, mimicking in vivo biofilm system so as to determine susceptibility of different phases of biofilm to antibiotics by three-dimensional analysis. Artificial mature biofilm of K. pneumoniae was made on black, polycarbonate membranes. Biofilm structure was visualized by scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM). Viable count method, CLSM and SEM analysis confirmed that mature, uniform and viable biofilms can be formed on the polycarbonate membranes by this method. The three-dimensional heterogeneity of biofilm was confirmed on the basis of results of CLSM, which is an important characteristics of in vivo biofilm system. Staining with the LIVE/DEAD BacLight viability kit and acridine orange suggested that the center of biofilm had more inactive cells compared with actively dividing cells on the periphery. Amikacin at a concentration of 40 μg ml−1 was effective against younger biofilm whereas ineffective against older biofilm that showed sparsely populated dead cells using the BacLight viability staining kit. Role of altered morphological characteristics toward increased antibiotic susceptibility was also studied for different phases of K. pneumoniae biofilm by CLSM and light microscopy. Thickness of biofilm increased from 0.093 to 0.231 mm with time. So, both heterogeneity and thickness of the biofilm are likely to influence the ineffectiveness of amikacin in older biofilm. The present model holds considerable clinical relevance and may be useful for evaluating the efficacy of antimicrobial agent on bacterial biofilms in vitro.
This work was supported by funding from the Department of Science and Technology (DST), India, in the form of a research fellowship. We thank Mr Deepak Bhatt of the Institute of Microbial Technology (IMTECH), Chandigarh, India for assistance in the operation of confocal microscope.