Covert dissemination of carbapenemase-producing Klebsiella pneumoniae (KPC) in a successfully controlled outbreak: long and short-read whole-genome sequencing demonstrate multiple genetic modes of transmission

Background: Carbapenemase-producing Enterobacteriaceae (CPE), including KPC-producing Klebsiella pneumoniae (KPC-Kpn), are an increasing threat to patient safety. Objectives: To use WGS to investigate the extent and complexity of carbapenemase gene dissemination in a controlled KPC outbreak. Materials and methods: Enterobacteriaceae with reduced ertapenem susceptibility recovered from rectal screening swabs/clinical samples, during a 3 month KPC outbreak (2013–14), were investigated for carbapenemase production, antimicrobial susceptibility, variable-number-tandem-repeat profile and WGS [short-read (Illumina), long-read (MinION)]. Short-read sequences were used for MLST and plasmid/Tn4401 fingerprinting, and long-read sequence assemblies for plasmid identification. Phylogenetic analysis used IQTree followed by ClonalFrameML, and outbreak transmission dynamics were inferred using SCOTTI. Results: Twenty patients harboured KPC-positive isolates (6 infected, 14 colonized), and 23 distinct KPC-producing Enterobacteriaceae were identified. Four distinct KPC plasmids were characterized but of 20 KPC-Kpn (from six STs), 17 isolates shared a single pKpQIL-D2 KPC plasmid. All isolates had an identical transposon (Tn4401a), except one KPC-Kpn (ST661) with a single nucleotide variant. A sporadic case of KPC-Kpn (ST491) with Tn4401a-carrying pKpQILD2 plasmid was identified 10 months before the outbreak. This plasmid was later seen in two other species and other KPC-Kpn (ST14,ST661) including clonal spread of KPC-Kpn (ST661) from a symptomatic case to nine ward contacts. Conclusions: WGS of outbreak KPC isolates demonstrated blaKPC dissemination via horizontal transposition (Tn4401a), plasmid spread (pKpQIL-D2) and clonal spread (K. pneumoniae ST661). Despite rapid outbreak control, considerable dissemination of blaKPC still occurred among K. pneumoniae and other Enterobacteriaceae, emphasizing its high transmission potential and the need for enhanced control efforts

Optimised long-read sequence bioinformatics tool for the analysis of the resistome and microbial communities

Public deliverable D-JRP12-2.1 & 2.2 'Optimised long-read sequence bioinformatics tool for the analysis of the resistome and microbial communities, publicly available' from FARMED EJP: Fast Antimicrobial Resistance and Mobile-Element Detection using metagenomics for animal and human on-site test

Review on current scientific literature and overview of commercially available methods for on-site DNA isolation

The development of tools for real-time detection of antimicrobial resistant (AMR) pathogens is a priority topic of the One Health EJP. For real-time analysis to be achievable on-site (away from the laboratory setting), robust culture independent detection methods, employing minimal equipment are required. Metagenomic sequencing using short-read data has provided insight and detailed compositions of a variety of microbial communities, as well as for the detection of potential pathogens and AMR or virulence genes. In addition to the currently bulky nature of short-read technologies and the difficulty to perform this analysis on site, an important limitation is their inability to reliably associate the genetic context of individual genes to bacteria (including pathogens) within a community. The FARMED project aims to address these issues by using the Oxford Nanopore Technologies (ONT) MinION, comparing to the current gold standard short-read technology, to evaluate its capability for diagnostic use on a range of sample matrices, particularly on-site at/near point of sample collection. This is enabled by the portability of the ONT technology, allowing on-site analysis, in contrast to short read sequencing. An additional advantage of using ONT sequencing or long-read metagenomic sequencing, is that the local genetic context of AMR genes can be derived, and as such, the presence of the AMR genes can be attributed to specific species or plasmids, within the bacterial community. This technology will enable the identification of a plethora of bacterial species and linkage of AMR genes to particular species. However, the successful application of on-site microbial detection/monitoring is influenced by various factors such as resource-limited working environment, sample collection and importantly the quality of the input DNA for sequencing. In addition, the availability of on-site sequencing and subsequent data analysis needs to be taken into account. This deliverable will review the scientific literature on existing DNA extraction methods and determine which have the potential/are suitable for rapid on-site metagenomic analysis. For on-site DNA extraction, it is essential that the implemented methods use minimal transportable equipment. Furthermore, the method(s) need to be free of hazardous chemicals for field personnel and the working environment, as well as components that require sub-zero transport conditions. We will also discuss the requirements of DNA for long-read sequencing and suitability of the different methods. We consider methods suitable for on-site DNA extraction from different ‘simple’ and ‘complex’ sample matrices, as each has different considerations. Finally, we will deliver recommendations for the FARMED consortium to be tested using ONT sequencing

Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches

This study aimed to assess the feasibility of using the Oxford Nanopore Technologies (ONT) MinION long-read sequencer in reconstructing fully closed plasmid sequences from eight Enterobacteriaceae isolates of six different species with plasmid populations of varying complexity. Species represented were Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens and Klebsiella oxytoca, with plasmid populations ranging from 1–11 plasmids with sizes of 2–330 kb. Isolates were sequenced using Illumina (short-read) and ONT’s MinION (long-read) platforms, and compared with fully resolved PacBio (long-read) sequence assemblies for the same isolates. We compared the performance of different assembly approaches including SPAdes, plasmidSPAdes, hybridSPAdes, Canu, Canu+Pilon (canuPilon) and npScarf in recovering the plasmid structures of these isolates by comparing with the gold-standard PacBio reference sequences. Overall, canuPilon provided consistently good quality assemblies both in terms of assembly statistics (N50, number of contigs) and assembly accuracy [presence of single nucleotide polymorphisms (SNPs)/indels with respect to the reference sequence]. For plasmid reconstruction, Canu recovered 70% of the plasmids in complete contigs, and combining three assembly approaches (Canu or canuPilon, hybridSPAdes and plasmidSPAdes) resulted in a total 78% recovery rate for all the plasmids. The analysis demonstrated the potential of using MinION sequencing technology to resolve important plasmid structures in Enterobacteriaceae species independent of and in conjunction with Illumina sequencing data. A consensus assembly derived from several assembly approaches could present significant benefit in accurately resolving the greatest number of plasmid structures

Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches

This study aimed to assess the feasibility of using the Oxford Nanopore Technologies (ONT) MinION long-read sequencer in reconstructing fully closed plasmid sequences from eight Enterobacteriaceae isolates of six different species with plasmid populations of varying complexity. Species represented were Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens and Klebsiella oxytoca, with plasmid populations ranging from 1–11 plasmids with sizes of 2–330 kb. Isolates were sequenced using Illumina (short-read) and ONT’s MinION (long-read) platforms, and compared with fully resolved PacBio (long-read) sequence assemblies for the same isolates. We compared the performance of different assembly approaches including SPAdes, plasmidSPAdes, hybridSPAdes, Canu, Canu+Pilon (canuPilon) and npScarf in recovering the plasmid structures of these isolates by comparing with the gold-standard PacBio reference sequences. Overall, canuPilon provided consistently good quality assemblies both in terms of assembly statistics (N50, number of contigs) and assembly accuracy [presence of single nucleotide polymorphisms (SNPs)/indels with respect to the reference sequence]. For plasmid reconstruction, Canu recovered 70% of the plasmids in complete contigs, and combining three assembly approaches (Canu or canuPilon, hybridSPAdes and plasmidSPAdes) resulted in a total 78% recovery rate for all the plasmids. The analysis demonstrated the potential of using MinION sequencing technology to resolve important plasmid structures in Enterobacteriaceae species independent of and in conjunction with Illumina sequencing data. A consensus assembly derived from several assembly approaches could present significant benefit in accurately resolving the greatest number of plasmid structures

Nano-imaging of intersubband transitions in van der Waals quantum wells

The science and applications of electronics and optoelectronics have been driven for decades by progress in growth of semiconducting heterostructures. Many applications in the infrared and terahertz frequency range exploit transitions between quantized states in semiconductor quantum wells (intersubband transitions). However, current quantum well devices are limited in functionality and versatility by diffusive interfaces and the requirement of lattice-matched growth conditions. Here, we introduce the concept of intersubband transitions in van der Waals quantum wells and report their first experimental observation. Van der Waals quantum wells are naturally formed by two-dimensional (2D) materials and hold unexplored potential to overcome the aforementioned limitations: They form atomically sharp interfaces and can easily be combined into heterostructures without lattice-matching restrictions. We employ near-field local probing to spectrally resolve and electrostatically control the intersubband absorption with unprecedented nanometer-scale spatial resolution. This work enables exploiting intersubband transitions with unmatched design freedom and individual electronic and optical control suitable for photodetectors, LEDs and lasers