A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae

Abstract

Enteric Gram-negative rods (GNR), which are frequent causes of community-acquired and nosocomial infections, are increasingly resistant to the antibiotics in our current armamentarium. One solution to this medical dilemma is the development of novel classes of antimicrobial compounds. Here we report the development of a robust, whole cell-based, high-throughput metabolic assay that detects compounds with activity against carbapenem-resistant Klebsiella pneumoniae. We have used this assay to screen approximately 8,000 fungal extracts and 50,000 synthetic compounds with the goal of identifying extracts and compounds active against a highly resistant strain of Klebsiella pneumoniae. The primary screen identified 43 active fungal extracts and 144 active synthetic compounds. Patulin, a known fungal metabolite and inhibitor of bacterial quorum sensing and alanine racemase, was identified as the active component in the most potent fungal extracts. We did not study patulin further due to previously published evidence of toxicity. Three synthetic compounds termed O06, C17, and N08 were chosen for further study. Compound O06 did not have significant antibacterial activity but rather interfered with sugar metabolism, while compound C17 had only moderate activity against GNRs. Compound N08 was active against several resistant GNRs and showed minimal toxicity to mammalian cells. Preliminary studies suggested that it interferes with protein expression. However, its direct application may be limited by susceptibility to efflux and a tendency to form aggregates in aqueous media. Rapid screening of 58,000 test samples with identification of several compounds that act on CR-K. pneumoniae demonstrates the utility of this screen for the discovery of drugs active against this highly resistant GNR.

Biosynthesis of Zinc Oxide Nanoparticles from Acacia nilotica (L.) Extract to Overcome Carbapenem-Resistant Klebsiella Pneumoniae

“…The UV visualization results showed a characteristic band of light absorption for zinc oxide nanoparticles in the range of 360–380 nm. This is similar to the results reported by Zare et al, who showed that the absorption peak for synthesized ZnO nanoparticles was in the range of 360–376 nm [ 19 ]. Other similar results were reported by Padalia et al and Safawo et al, who found that ZnO-NPs synthesized using A. hydrophila and O. europea extract showed an absorption peak 374 nm.…”

Effects of Zinc Oxide Nanoparticles Synthesized Using Aspergillus niger on Carbapenem-Resistant Klebsiella pneumonia In Vitro and In Vivo

“…In Saudi Arabia, the high incidence of KPC may be due to the large number of pilgrims, visitors, and migrant workers from endemic countries such as Turkey, India, and Pakistan every year ( Liao et al., 2018 ). Surveys such as that conducted by Faiz and Khan have shown that the incidence of KPC among Makkah hospitals is about 48.4% from other carbapenem-producing organisms ( Aftab Faiz, 2016 ).…”

High-Throughput Screening of Natural Product and Synthetic Molecule Libraries for Antibacterial Drug Discovery

“…Genetic manipulation has also been utilized to help finding molecular targets of antibacterial agents, such as sensitive-resistant pair screening (philipimycin), target upregulation or overproduction and gain of the control rescue assay (novobiocin), reduced expression of target or hypersensitive screening (cerulenin), etc. Traditional CT-HTS and MT-HTS assays using either natural products or synthetic molecule libraries have generally been more successful in identifying anti-gram-positive antibacterial agents than anti-gram-negative antibacterial agents with some exceptions [ 49 ]. However, HTS using multicopy suppressor containing the genomic library of E. coli demonstrated successful identification of antibiotic targets in addition to demonstrating the roles of multidrug efflux pump in reducing the success of antibiotics, a major problem for gram-negative bacteria [ 50 ], which in general has a hit rate of 1–2 reduced orders of magnitude more than gram-positive, S. aureus [ 33 ].…”