Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity

Abstract

The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil‐derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole‐genome sequencing and linker‐mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin‐resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization.

Bacterial analogs of plant piperidine alkaloids mediate microbial interactions in a rhizosphere model system

“…Another Pseudomonas isolate (SWI36) was reported to inhibit B. cereus , and its activity was dependent on a koreenceine-type gene cluster from clade B (18), but we found that it did not inhibit F. johnsoniae . Under certain conditions, P. koreensis inhibited B. cereus , and the activity was dependent on the koreenceine cluster, suggesting a similarity with the SWI36 cluster (Fig.…”

“…Similar koreenceine-like clusters have previously been identified by functional screens for antimicrobial activities (17, 18); however, there are no reports of the metabolites produced. We identified 179 koreenceine-like clusters in genomes in NCBI (June 2018).…”

“…We identified a gene cluster necessary for the production of the koreenceine metabolites. Other koreenceine-like clusters have been predicted to mediate (22) and others are associated with (17, 18) antagonistic activity against diverse microorganisms. Thirty-five percent of P. koreensis and P. mandelii genomes in NCBI contain this cluster and there are at least 160 Pseudomonas genomes harboring a related cluster, indicating the widespread nature of this cluster among Pseudomonas spp.…”

Fitting the message to the location: engaging adults with antimicrobial resistance in a World War 2 air raid shelter

“…Children are the target audience for games such as ebug (McNulty et al 2011), plays (https://www.microbiolo gysociety.org/event/education-outreach-events/stopping-thespread-of-superbugs-2012.html) and hands-on activities such as the Small Worlds Initiative (Davis et al 2017) and Antibiotics Unearthed (https://www.microbiologysoc iety.org/education-outreach/antibiotics-unearthed.html). At family-focused events, adults tend to work with the scientists to engage their own children.…”

Why is Scientific Research on ‘Data-Poor’ Microorganisms Being Ignored?

“…Importantly, although it is tempting to think of the added value of data-poor microorganisms as being restricted to research and publications involving comparative genome sequencing, there actually exist many more possibilities for research involving data-poor microorganisms. These include (to name only a few) -pathogenicity studies: investigating the patho genicity of historically significant microorganisms [6], discovery of variation in (unknown) mobile genetic elements [7], discovery of variation in (unknown) virulence factors and plasmids [8], identification of new antibiotic/antiviral/ antifungal/antiparasitic mechanisms and anti biotics [9], investigating antibiotic 'tolerance' [10], transformation, transduction, conjugation efficiencies [11]; diagnosis and treatment: development of new diagnostic tests and targets [12], discovery of previously unknown antibiotics [13], investigating microorganisms and cancer therapy [14], extended standardization of genotyping schemes [15]; gene regulation: investigating global gene regulation, for example, SOS and stringent responses [16], linking transcription factors to global physiology [17], reconstruction and prediction of transcription regulatory networks [18]; evolution: evolutionary ecological transitions [19]; and the immune system: differences in inflammatory responses [20].…”