Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.

Objective: With a rise in multidrug resistant (MDR) bacterial isolates, search for antibiotics or compounds that could act synergistically with them is a significant area of research. Efflux-mediated resistance, in particular, is a great hurdle that needs to be overcome. In an effort to identify such synergistic compounds and potential efflux pump inhibitors (EPI), we analyzed the rind of Punica granatum (pomegranate) against MDR clinical Klebsiella pneumoniae isolates.Methods: Sequential fractionation of P. granatum rind ethanol (PGR) extract was carried out to obtain hexane, butanol and water fractions. Antibacterial activity of the plant extracts was confirmed, and synergistic interaction with antibiotics was determined by the checkerboard assay. Gas chromatography-mass spectrometry (GC-MS) analysis was performed to identify the phytochemical constituents of the hexane extract. To study EPI activity of the extracts, norfloxacin accumulation assay was carried out.Results: PGR ethanol extract was found to have synergistic activity with ciprofloxacin, levofloxacin, ceftazidime, cefoxitin, meropenem, and gentamicin resulting in fold decrease of minimum inhibitory concentration (MIC) ranging from 2 to 32 fold. The hexane fraction was found to have maximum synergistic activity resulting in a 32-fold reduction of ciprofloxacin MIC followed by butanol and water fractions. The PGR ethanol extract was also found to have efflux inhibition activity by the norfloxacin accumulation assay. Of the sequential fractions, the butanol fraction had maximum efflux inhibition activity.Conclusion: Therefore, our study shows that PGR extract can potentiate the effect of antibiotics on MDR bacteria, and the mode of action is likely to be due to EPI.

A new pyrrolidine alkaloid named Punigratane was isolated from the rind of Punica granatum. This is the first report of a pyrrolidine-like structure from the rind. The activity of this compound was tested in a representative MDR Klebsiella pneumoniae strain which exhibited high efflux pump activity. At a concentration of 6 mg, this compound Punigratane was found to have efflux inhibition activity.

Objective: Efflux-mediated resistance is a growing therapeutic complication as it reduces the efficacy of antibiotics. In gram-negative bacteria like E. coli and K. pneumoniae, this can be overcome with the help of efflux pump inhibitors (EPI) targeted at the transporter protein AcrB that plays a key role in binding to antibiotics. Our study focuses on the potential EPI Punigratane isolated from the rind of Punica granatum. Using computational docking analysis and in silico analysis, our aim is to determine whether Punigratane has the ability to interact and inhibit the AcrB pump and whether it has drug viability.Materials: Computational docking analyses were carried out using the online platforms Mcule and PatchDock. Drug-likeness and classification of Punigratane was predicted using online tools PreADMET and SuperPred. Admet SAR and Toxicity Checker at Mcule were used to predict ADME (absorption, distribution, metabolism, and excretion) and overt toxicity properties.Results: Punigratane was computationally docked with 57 AcrB crystal structures available at the PDB database to determine whether it could bind to the active site regions. It was found to bind in the periplasmic region close to the substrate bile acid where it is thought to bring about inhibition by steric hindrances. When docked with AcrB mutant (AcrB N109A), it was found to bind in the same periplasmic site as the substrates (EtBr, Rhodamine 6G, Ciprofloxacin, Bile acid) as well as the inhibitor (phenylalanine-arginine β-naphthylamide-PaβN). When docked in the active site of the inhibitor MBX2319, it was found to have a comparable docking score as well as the same hydrophobic interactions as the inhibitor. In silico analysis showed that Punigratane exhibited a drug-likeness to the inhibitor MBX2319 and that its drug classification is similar to antimicrobial agents. It was also found be a potential drug due to its intestinal absorption, increased bioavailability and non-toxic nature.Conclusion: Therefore our report shows that Punigratane could be a potential drug candidate that inhibits efflux activity by interacting and inhibiting the AcrB efflux pump.