Optimization and Screening of entB and uge genes in the clinical isolates of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is listed as an ESKAPE pathogen by the World Health Organization (WHO) and has been recently associated with higher morbidity and mortality. K. pneumoniae exhibits multidrug resistance mechanisms which is influenced by various factors that includes the mutations within the ribosomal binding site, acquisition of mobile genetic elements carrying resistance genes. Along with the presence of other types of resistance mechanisms, one is the ribosomal protection and biofilm formation exemplified by the uge and entB genes. There were 156 isolates of K. pneumoniae in all, collected from tertiary care hospitals in Islamabad, Pakistan. So, this study focused on the phenotypic analysis and genotypic screening of uge and entB genes in K. pneumoniae. Gram staining and colony morphology were used to determine morphological characteristics. Following that, biochemical identification was accomplished through a series of assays. Further, the doxycycline on planktonic and biofilm of K. pneumoniae was tested. This study involved the use of Minimum Biofilm Eradication Concentration (MBEC), Minimum Biofilm Inhibition Concentrations (MBIC), and sub minimal concentration of doxycycline on isolates of K. pneumoniae. Screening of 156 isolates revealed that 38% isolates carried uge gene which encodes for enzyme UDP glucose 4 epimerases involved in extracellular polysaccharides synthesis and 13% isolates carried entB gene (Enterobactin synthase component B) which is used to scavenge iron to provide characteristic role in biofilm formation. While 14 out of 156 total isolates (9%) carried both genes. Then confirmatory tests including disk diffusion and minimum inhibitory concentration were performed. Our disk diffusion results showed that 11 out of 80 (11%) were doxycycline resistant isolates while 24 out of 80 (30%) were doxycycline sensitive isolates. Further, confirmatory tests to determine the minimal inhibitory doses for doxycycline includes MIC-p and MIC-b measurements. The results were found to be almost same as disk diffusion results i.e., 24 out of 30 (30%) isolates were found sensitive at MIC value ranged from 1-4ug/ml. Further, biofilm forming capacity was determined by microtiter plate assay. We identified two strong biofilm formers, one moderate biofilm former and five weak biofilm former isolates. Biofilms are generally associated with the increase in virulence and resistance of bacteria as our study indicated increase in MBEC (512-128ug/ml) and MBIC (128-32ug/ml) value of ix doxycycline after biofilm formation. Further, this study indicated an increase in the biofilm forming ability of isolates with time when exposed to doxycycline at sub minimal concentrations. Hence, our findings indicated a greater susceptibility of K. pneumoniae to doxycycline in biofilm form.