Utilization of Vibrio cholerae as a Model Organism to Screen Natural Product Libraries for Identification of New Antibiotics

Abstract

The development of antibiotic-resistant bacteria requires increasing research efforts in drug discovery. Vibrio cholerae can be utilized as a model gram-negative enteric pathogen in high- and medium-throughput screening campaigns to identify antimicrobials with different modes of action. In this chapter, we describe methods for the optimal growth of V. cholerae in 384-well plates, preparation of suitable microtiter natural product sample libraries, as well as their screening using measurements of bacterial density and activity of type II secretion-dependent protease as readouts. Concomitant LC-MS/MS profiling and spectral data networking of assay sample libraries facilitate dereplication of putative known and/or nuisance compounds and efficient prioritization of samples containing putative new natural products for further investigation.

Chlorination (but Not UV Disinfection) Generates Cell Debris that Increases Extracellular Antibiotic Resistance Gene Transfer via Proximal Adsorption to Recipients and Upregulated Transformation Genes

“…The natural transformation of eARGs was conducted using competent V. vulnificus as the recipient. ,,, Briefly, the natural competence of V. vulnificus was prepared using a chitin-based assay. , Adsorbed eARGs (for chlorination) and free eARGs (for UV irradiation) were prepared as described above.…”

Exploring the Potential of Antibiotic Production From Rare Actinobacteria by Whole-Genome Sequencing and Guided MS/MS Analysis

“…For instance, MALDI-TOF-MS was used to identify 10 secondary metabolites related to previously uncharacterized gene clusters in Streptomyces hygroscopicus (Kersten et al, 2011), ESI-IT-MS was applied to uncover a compound of strong antibacterial activity produced by Gliocladium sp. (Koolen et al, 2012), and a Q-Trap LC-MS platform was utilized to unravel natural products with antimicrobial activity produced by Vibrio cholerae (Sikora et al, 2018). Therefore, the coupled method of genome mining and mass spectrometry could reveal the antibiotic production potential of isolated microbes.…”