Digital multiplex ligation assay for highly multiplexed screening of β-lactamase-encoding genes in bacterial isolates

Increasing incidence of antibiotic resistance in clinical and environmental settings calls for increased scalability in their surveillance. Current screening technologies are limited by the number of samples and genes that can easily be screened. We demonstrate here digital multiplex ligation assay (dMLA) as a low-cost targeted genomic detection workflow capable of highly-parallel screening of bacterial isolates for multiple target gene regions simultaneously. Here, dMLA is used for simultaneous detection of 1187 β-lactamase-encoding genes, including extended spectrum β-lactamase (ESBL) genes, in 74 bacterial isolates. We demonstrate dMLA as a light-weight and cost-efficient workflow which provides a highly scalable tool for antimicrobial resistance surveillance and is also adaptable to genetic screening applications beyond antibiotic resistance.

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced β-galactosidase production using the CRISPR/Cas9 system

The ganA gene from Bacillus subtilis encoding a β-galactosidase for degradation of the galactomannan was integrated in different loci of the B. subtilis chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of ganA cassettes in which the ganA gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of ganA expression cassette showed a β-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of β-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of ganA in the chromosome produced β-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.