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In light of the limited therapeutic options and significant threat associated with infections caused by Carbapenem Resistant Enterobacteriaceae (CRE) pathogens, understanding the pathogensâ biology, specifically, the ability of form the biofilms and biofilm related gene expression are utmost important to the treatment of the biofilm-associated infections caused by these CRE pathogens. To understand the pathogenic role of the biofilms, the study investigated amount of biofilm formation and biofilm-related gene expression in in CRE strains, Escherichia coli IMP-type and Klebsiella pneumoniae NDM- 1. The amount of biofilm formed was measured using Tissue Culture plate assay at different time points (6, 12, 24 and 48 hours) of incubation, assuming the time points were corelate with the stages of biofilm development stages; initial attachment (6 hours), microcolony formation (12 hours), maturation of biofilms (24 hours) and dispersion of biofilm (48 hours). Biofilms were quantified under static and shaking incubator conditions and under two different growth media; nutrient poor (AB broth) and nutrient rich media (LB broth) media. In parallel, Quantitative Real-time PCR (qPCR) was applied for invitro biofilm development stages. The amount of biofilms formed and the biofilm-related gene expression results were compared with structural analysis using Confocal Laser Scanning Microscopy (CLSM) at different growth conditions. For both CRE strains irrespective of incubation condition and growth media, varying amount of biofilm was observed and was corelated with the biofilm development stages. Maximum amount of biofilm development was observed at 24 hour time period of incubation indicating the maturation of biofilm development. Similarly, the same pattern of biofilm-related gene expression was observed, where majority of gene expressed at 24 hour time point. In related to the above analysis CLSM also, showed that aggregation of the different cellular products, corresponding to each biofilm development stage. These findings suggested that, for both CRE organismsâ biofilm formation and biofilm-related gene expression profiles are not affected by the various growth conditions tested, indicating the successful pathogenic role of biofilm formation by adopting to different environmental conditions. The findings suggest that adoptability of biofilm under different environmental conditions plays a significant role in the carbapenem-resistant Escherichia coli IMP-type and Klebsiella pneumoniae NDM-1 bacteria in their increased prevalence in biofilm-associated infections.