The need for novel diagnostic techniques that can rapidly guide effective treatment is, therefore, urgent. Especially troublesome are Gram-negative bacilli encoding plasmid-borne antibiotic resistance genes, some of which are on the WHO antimicrobial resistance priority pathogen list 3. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management.īacterial infections cause millions of deaths every year 1, 2, and the number is expected to rise due to the increase in antibiotic resistance, making treatment more challenging. The optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids. The single-molecule aspect of the method enables us to identify multiple E. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. At a taxonomic resolution corresponding to E. We demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization.

In this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions. Identification of pathogens is crucial to efficiently treat and prevent bacterial infections.

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