Optimization and Screening of WabG and AcrAB genes in the clinical isolates of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae causes a variety of infections including conununity-acquired pneumonia, hospital-associated infections, and urinary tract infections. It has garnered attention from the World Health Organization (WHO) due to its ranking among the top three pathogens exhibiting multidrug resistance. This resistance is influenced by various factors, including the acquisition of mobile genetic elements carrying specific resistance genes, mutations within the ribosomal binding site, and clu-omosomal mutations leading to increased resistance. Other mechanisms involve the efflux system, exemplified by the acrAB gene, and the formation of biofilms, driven by the wabG gene. This study is focused on screening the acrAB and wabG genes in K. pneumoniae. Further, we investigated doxycycline on the planktonic and biofilm of K. pneumoniae. We investigated Minimum Biofilm Eradication Concentration (MBEC), Minimum Biofilm Inhibitory Concentration (MBIC), and sub-MIC concentrations of doxycycline on K. pneumoniae isolates. Screening of n= 15 6 isolates revealed occurrence of 33% for the acrAB gene (Acriflavine resistance channel protein A and B) which encodes for AcrA and AcrB proteins that act as the periplasmic adaptor protein (PAP) in several RND tripartite efflux pumps, and as a proton-drug antiporter respectively to provide characteristic intrinsic resistance to antimicrobials and 25% for the wabG gene which is involved in attacmnent to a-I-glycero-d-matmo-heptopyranose II (l,d-HeppII) at the 0 - 3 position of an a-d-galactopyranosyluronic acid (a-d-GaIAp) residue. The co occurrence of these genes was fOlmd to be 8%. We tested doxycycline for MIC-p and MIC-b measurements. Sensitivity testing against planktonic cells indicated MIC values ranged from 1-4flg/ml. We assessed biofilm-forming capacity by microtiter plate assay and identified two strong biofilm formers. Biofilms are generally associated with higher levels of virulence and resistance which was reflected in MBEC (512-256~lg/ml) and MBIC (l28-64~lg/ml) in this study. At subminimal concentrations, a reduction in the biofilm-forming ability of the selected isolates was also observed. The most effective doxycycline concentration against our K. pnemoniae isolates showed to be 5~lg/ml. Our results confirm a higher tolerance of K. pneumoniae to doxycycline in biofilm form.