Combining isolation chips with velvet transfer for high-throughput primary screening of antibiotic-producing soil-dwelling bacteria

Abstract

Antimicrobial resistance (AMR) remains a significant threat to global health, requiring various mitigation strategies. Soil-dwelling microbes have long been a valuable source of novel antibiotics; however, their low cultivability using standard methods has often led to the discovery of known antibiotics. This bottleneck has been partially overcome by using isolation chips (iChips), which enhance the cultivability (both in terms of number and diversity) of soil bacteria. This has created a need to find new screening methods that allow high-throughput screening, are less resource-intensive, and can screen multiple test organisms. To address this, soil samples were collected from three different regions of Tanzania, and numerous bacterial soil species colonies were grown in iChips and then transferred to master plates. Replica plates were created using a velvet transfer method and utilized for parallel agar overlay assays against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Master plates with 33-62 colonies showing variation in size, morphology, and spatial arrangement were produced by cultivating agar plugs from iChips. Three to four replicates, which preserved these characteristics at the transfer efficiencies of 91.3-100% were obtained without contamination. Zones of inhibition -to-colony diameter ratios ranging 1.15-3.27 were obtained following parallel primary screening against test strains, hence enabling the identification of potential antibiotic producers. This is the first report of the combined iChip and velvet transfer workflow that offers a high-throughput, resource-efficient, and scalable method for primary screening of various soil-derived bacteria for antibacterial Natural Products (NPs), while also maintaining the master plates for downstream investigations.