Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples

Facilitated sequence assembly using densely labeled optical DNA barcodes:A combinatorial auction approach

<div><p>The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard “foreign” contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.</p></div

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Detailed characterization of plasmids carrying resistance genes using optical DNA mapping

We present an assay, based on optical DNA mapping in nanochannels that is capable of characterizing the plasmid content of bacterial isolates resistant to antibiotics in a fast an detailed way. In a single experiment we determine the number of different plasmids in each sample, their size, as well as a barcode that can be used for plasmid identification and tracing. In addition we demonstrate that we can identify resistance genes on individual plasmids using CRISPR/Cas9. We foresee that the assay can be a useful tool all the way from fundamental plasmid biology to diagnostics and surveillance of resistant infections

Detailed characterization of plasmids carrying resistance genes using optical DNA mapping

We present an assay, based on optical DNA mapping in nanochannels that is capable of characterizing the plasmid content of bacterial isolates resistant to antibiotics in a fast an detailed way. In a single experiment we determine the number of different plasmids in each sample, their size, as well as a barcode that can be used for plasmid identification and tracing. In addition we demonstrate that we can identify resistance genes on individual plasmids using CRISPR/Cas9. We foresee that the assay can be a useful tool all the way from fundamental plasmid biology to diagnostics and surveillance of resistant infections

Rapid tracing of resistance plasmids in a nosocomial outbreak using optical DNA mapping

Resistance to life-saving antibiotics increases rapidly worldwide, and multiresistant bacteria have become a global threat to human health. Presently, the most serious threat is the increasing spread of Enterobacteriaceae carrying genes coding for extended spectrum β-lactamases (ESBL) and carbapenemases on highly mobile plasmids. We here demonstrate how optical DNA maps of single plasmids can be used as fingerprints to trace plasmids, for example, during resistance outbreaks. We use the assay to demonstrate a potential transmission route of an ESBL-carrying plasmid between bacterial strains/species and between patients, during a polyclonal outbreak at a neonatal ward at Sahlgrenska University Hospital (Gothenburg, Sweden). Our results demonstrate that optical DNA mapping is an easy and rapid method for detecting the spread of plasmids mediating resistance. With the increasing prevalence of multiresistant bacteria, diagnostic tools that can aid in solving ongoing routes of transmission, in particular in hospital settings, will be of paramount importance

Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules

The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics

Contig scaffolding using Illumina contigs from a mixed sample of pUUH/chromosomal DNA.

<p>(Top) Optimal placement of the contig theory barcodes on the experimental pUUH barcode using our contig scaffolding method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193900#sec002" target="_blank">Methods</a>). 220 contigs were obtained through Illumina sequencing of a mixed sample containing the pUUH plasmid and chromosomal DNA from the bacterium <i>Klebsiella pneumoniae</i>. Based on a sequence alignment 16 of the contigs are deemed to belong to the pUUH plasmid. Horizontal lines at the top corresponds to “true” contig positions based on a sequence comparison of the full pUUH sequence and the contig sequences. We find that 2 contig barcodes pass the length and p-value thresholds. The two contigs which were placed ended up at correct positions. (Bottom) The examples of removed contigs illustrates intensity profiles of a few typical non-matching barcodes: the four chromosomal contig barcodes with the smallest p-values and the third longest plasmid barcode (orange).</p

Contig scaffolding using synthetic contigs from a mixed sample of pUUH/chromosomal DNA.

<p>Synthetic contigs were generated by randomly cutting the known DNA sequences for pUUH and the chromosomal DNA from <i>Klebsiella pneumoniae</i>. The distances between cuts were taken from a truncated exponential distribution. We then applied our contig scaffolding method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193900#sec002" target="_blank">Methods</a>). (Top) Three typical examples of contig barcodes assembled on the consensus pUUH barcode, here with average contig size = 24.5 kbps. In the first two examples all placed contigs end up at correct positions, whereas in the third example there is one misplaced contig barcode. (Bottom) The two ratios: the filling fraction = number of occupied pixels/total number of pixels, and the number of correctly placed contigs/total number of contigs were calculated. This was repeated for 100 random realizations of the cutting process, and mean values and associated standard deviations were calculated. We find that our method is effective at separating chromosomal and pUUH DNA and, also, it rarely places a contig at the wrong position. The filling fraction increases with increasing contig size. A similar plot for the p4_2_1.1 plasmid is found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193900#pone.0193900.s013" target="_blank">S11 Fig</a>.</p

Contig scaffolding using synthetic contigs from a pure pUUH contig sample (no chromosomal DNA).

<p>Synthetic contigs were generated by randomly cutting the known DNA sequence for the pUUH plasmid. The distances between cuts were taken from a truncated exponential distribution with average sizes varying from 10 kbps to 80 kbps. We then applied our contig scaffolding method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193900#sec002" target="_blank">Methods</a>). (Top) Example of contig barcodes assembled on the consensus pUUH barcode, here with average contig size = 24.5 kbps. (Bottom) Two placement ratios: the filling fraction = number of occupied pixels/total number of pixels in experimental barcode, and correct placement ratio = number of correctly placed contigs/total number of contigs. This was repeated for 100 random realizations of the cutting process, and mean values and associated standard deviations for these ratios were calculated. A similar plot for the p4_2_1.1 plasmid is found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193900#pone.0193900.s010" target="_blank">S8 Fig</a>.</p