PUNICA GRANATUM RIND EXTRACT: ANTIBIOTIC POTENTIATOR AND EFFLUX PUMP INHIBITOR OF MULTIDRUG RESISTANT KLEBSIELLA PNEUMONIAE CLINICAL ISOLATES

ABSTRACTObjective: With a rise in multidrug resistant (MDR) bacterial isolates, search for antibiotics or compounds that could act synergistically with themis a significant area of research. Efflux-mediated resistance, in particular, is a great hurdle that needs to be overcome. In an effort to identify suchsynergistic compounds and potential efflux pump inhibitors (EPI), we analyzed the rind of Punica granatum (pomegranate) against MDR clinicalKlebsiella 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. Gaschromatography-mass spectrometry (GC-MS) analysis was performed to identify the phytochemical constituents of the hexane extract. To study EPIactivity 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 gentamicinresulting in fold decrease of minimum inhibitory concentration (MIC) ranging from 2 to 32 fold. The hexane fraction was found to have maximumsynergistic activity resulting in a 32-fold reduction of ciprofloxacin MIC followed by butanol and water fractions. The PGR ethanol extract was alsofound to have efflux inhibition activity by the norfloxacin accumulation assay. Of the sequential fractions, the butanol fraction had maximum effluxinhibition 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 tobe due to EPI.Keywords: Punica granatum rind, Pomegranate, Synergy with antibiotics, Multidrug resistant, Klebsiella pneumoniae, Efflux pump inhibition

Peptide-based direct electrochemical detection of receptor binding domains of SARS-CoV-2 spike protein in pristine samples

RNA isolation and amplification-free user-friendly detection of SARS-CoV-2 is the need of hour especially at resource limited settings. Herein, we devised the peptides of human angiotensin converting enzyme-2 (hACE-2) as bioreceptor at electrode interface for selective targeting of receptor binding domains (RBD) of SARS-CoV-2 spike protein (SP). Disposable carbon-screen printed electrode modified with methylene blue (MB) electroadsorbed graphene oxide (GO) has been constructed as cost-efficient and scalable platform for hACE-2 peptide-based SARS-CoV-2 detection. In silico molecular docking of customized 25 mer peptides with RBD of SARS-CoV-2 SP were validated by AutoDock CrankPep. N-terminal region of ACE-2 showed higher binding affinity of − 20.6 kcal/mol with 15 H-bond, 9 of which were < 3 Å. Electrochemical biosensing of different concentrations of SPs were determined by cyclic voltammetry (CV) and chronoamperometry (CA), enabling a limit of detection (LOD) of 0.58 pg/mL and 0.71 pg/mL, respectively. MB-GO devised hACE-2 peptide platform exert an enhanced current sensitivity of 0.0105 mA/pg mL(−1) cm(−2) (R(2) = 0.9792) (CV) and 0.45 nA/pg mL(−1) (R(2) = 0.9570) (CA) against SP in the range of 1 pg/mL to 1 µg/mL. For clinical feasibility, nasopharyngeal and oropharyngeal swab specimens in viral transport medium were directly tested with the prepared peptide biosensor and validated with RT-PCR, promising for point-of-need analysis

Antibiotherapy and pathogenesis of uncomplicated UTI: difficult relationships

In a time when conventional antibiotics are becoming increasingly less effective for treatment of infections, the relationship between bacteria and antimicrobial resistance is becoming more and more complicated. This paper provides a current review of studies reported in the literature pertaining to the antibiotherapy of human urinary tract infections (UTI), in a way that helps the reader direct a bibliographic search and develop an integrated perspective of the subject. Highlights are given to (bio)pathogenesis of uncomplicated cystitis. Features associated with the antibiotherapy of UTI such as development of resistance are presented in the text systematically. This review discusses recent advances in the understanding of how the predominant uropathogen Escherichia coli interacts with its host and leads to infection; so one can understand some of the reasons behind antibiotherapy failures

COMPUTATIONAL DOCKING AND IN SILICO ANALYSIS OF POTENTIAL EFFLUX PUMP INHIBITOR PUNIGRATANE

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.Â

COMPUTATIONAL DOCKING AND IN SILICO ANALYSIS OF POTENTIAL EFFLUX PUMP INHIBITOR PUNIGRATANE

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.Â

A global comprehensive study of the distribution of type I-E and type I-E* CRISPR-Cas systems in Klebsiella pneumoniae

Background: The CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems are the short DNA sequences and RNA-dependent nuclease involved in the adaptive immunity in bacteria and archaea. The type of CRISPR-Cas system influences antibiotic susceptibility in Klebsiella pneumoniae. Here, our objective was to study the diversity of CRISPR-Cas system in the genome of K. pneumoniae from the available whole genome sequencing (WGS) data. Material and Methods: We identified the CRISPR-Cas systems of K. pneumoniae using the CRISPR-CasFinder database. The complete genome sequence and its submission details were obtained from the National Center for Biotechnology Information (NCBI) database. Results: A total of 1607 K. pneumoniae whole genome sequences were analyzed. The major contributors of WGS data of K. pneumoniae were China (26.6%), United States (21.5%), Australia (10%), South Korea (8%), India (5.5%), and United Kingdom (4.9%). Out of 1607 genomes analyzed, almost one-fourth were CRISPR-Cas positive (403/1607) and three-fourth were CRISPR-Cas negative (1204/1607). Among CRISPR-Cas positive strains, 220 belonged to type I-E* and 183 were type I-E. Furthermore, type I-E* CRISPR-Cas systems were significantly higher in Asia (P < 0.001), whereas type I-E were significantly higher in Europe (P < 0.01). Among countries, typically, type I-E* strains were found to be higher in China (P < 0.01) and India (P < 0.01), whereas type I-E strains were higher in Germany (P < 0.01). Conclusion: Hence, it is important to know the type of CRISPR-Cas systems in K. pneumoniae strains across the countries and it can help to understand the diversity of CRISPR-Cas systems worldwide

Punigratane, a novel pyrrolidine alkaloid from <i>Punica granatum</i> rind with putative efflux inhibition activity

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