Methylation analysis of Klebsiella pneumoniae from Portuguese hospitals.

Klebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden. The application of long read sequencing technologies is providing insights into bacterial chromosomal and putative extra-chromosomal genetic elements (PEGEs) associated with AMR, but also epigenetic DNA methylation, which is thought to play a role in cleavage of foreign DNA and expression regulation. Here, we apply the PacBio sequencing platform to eight Portuguese hospital isolates, including one carbapenemase producing isolate, to identify methylation motifs. The resulting assembled chromosomes were between 5.2 and 5.5Mbp in length, and twenty-six PEGEs were found. Four of our eight samples carry blaCTX-M-15, a dominant Extended Spectrum Beta Lactamase in Europe. We identified methylation motifs that control Restriction-Modification systems, including GATC of the DNA adenine methylase (Dam), which methylates N6-methyladenine (m6A) across all our K. pneumoniae assemblies. There was a consistent lack of methylation by Dam of the GATC motif downstream of two genes: fosA, a locus associated with low level fosfomycin resistance, and tnpB transposase on IncFIB(K) plasmids. Overall, we have constructed eight high quality reference genomes of K. pneumoniae, with insights into horizontal gene transfer and methylation m6A motifs.

Whole genome sequencing of amplified Plasmodium knowlesi DNA from unprocessed blood reveals genetic exchange events between Malaysian Peninsular and Borneo subpopulations.

The zoonotic Plasmodium knowlesi parasite is the most common cause of human malaria in Malaysia. Genetic analysis has shown that the parasites are divided into three subpopulations according to their geographic origin (Peninsular or Borneo) and, in Borneo, their macaque host (Macaca fascicularis or M. nemestrina). Whilst evidence suggests that genetic exchange events have occurred between the two Borneo subpopulations, the picture is unclear in less studied Peninsular strains. One difficulty is that P. knowlesi infected individuals tend to present with low parasitaemia leading to samples with insufficient DNA for whole genome sequencing. Here, using a parasite selective whole genome amplification approach on unprocessed blood samples, we were able to analyse recent genomes sourced from both Peninsular Malaysia and Borneo. The analysis provides evidence that recombination events are present in the Peninsular Malaysia parasite subpopulation, which have acquired fragments of the M. nemestrina associated subpopulation genotype, including the DBPß and NBPXa erythrocyte invasion genes. The NBPXb invasion gene has also been exchanged within the macaque host-associated subpopulations of Malaysian Borneo. Our work provides strong evidence that exchange events are far more ubiquitous than expected and should be taken into consideration when studying the highly complex P. knowlesi population structure.

Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research.

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

Whole-genome sequencing resolves a polyclonal outbreak by extended-spectrum beta-lactam and carbapenem-resistant Klebsiella pneumoniae in a Portuguese tertiary-care hospital.

Klebsiella pneumoniae has emerged as an important nosocomial pathogen, with whole-genome sequencing (WGS) significantly improving our ability to characterize associated outbreaks. Our study sought to perform a genome-wide analysis of multiclonal K. pneumoniae isolates (n=39; 23 patients) producing extended spectrum beta-lactamases and/or carbapenemases sourced between 2011 and 2016 in a Portuguese tertiary-care hospital. All isolates showed resistance to third-generation cephalosporins and six isolates (five patients) were also carbapenem resistant. Genome-wide-based phylogenetic analysis revealed a topology representing ongoing dissemination of three main sequence-type (ST) clades (ST15, ST147 and ST307) and transmission across different wards, compatible with missing links that can take the form of undetected colonized patients. Two carbapenemase-coding genes were detected: blaKPC-3, located on a Tn4401d transposon, and blaGES-5 on a novel class 3 integron. Additionally, four genes coding for ESBLs (blaBEL-1, blaCTX-M-8, blaCTX-M-15 and blaCTX-M-32) were also detected. ESBL horizontal dissemination across five clades is highlighted by the similar genetic environments of blaCTX-M-15 gene upstream of ISEcp1 on a Tn3-like transposon. Overall, this study provides a high-resolution genome-wide perspective on the epidemiology of ESBL and carbapenemase-producing K. pneumoniae in a healthcare setting while contributing for the adoption of appropriate intervention and prevention strategies.

Structural insights into the human metapneumovirus glycoprotein ectodomain.

Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral attachment glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering, and small-angle X-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, intrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.