Emergence of OXA-48-producing Klebsiella pneumoniae in Lithuania, 2023: a multi-cluster, multi-hospital outbreak.

In 2023, an increase of OXA-48-producing Klebsiella pneumoniae was noticed by the Lithuanian National Public Health Surveillance Laboratory. Whole genome sequencing (WGS) of 106 OXA-48-producing K. pneumoniae isolates revealed three distinct clusters of carbapenemase-producing K. pneumoniae high-risk clones, including sequence type (ST) 45 (n = 35 isolates), ST392 (n = 32) and ST395 (n = 28), involving six, six and nine hospitals in different regions, respectively. These results enabled targeted investigation and control, and underscore the value of national WGS-based surveillance for antimicrobial resistance.

Emergence and persistent spread of carbapenemase-producing Klebsiella pneumoniae high-risk clones in Greek hospitals, 2013 to 2022.

BackgroundPreliminary unpublished results of the survey of carbapenem- and/or colistin-resistant Enterobacterales (CCRE survey) showed the expansion of carbapenemase-producing Klebsiella pneumoniae (CPKP) sequence type (ST) 39 in 12 of 15 participating Greek hospitals in 2019.AimWe conducted a rapid survey to determine the extent of spread of CPKP high-risk clones in Greek hospitals in 2022 and compare the distribution of circulating CPKP clones in these hospitals since 2013.MethodsWe analysed whole genome sequences and epidemiological data of 310 K. pneumoniae isolates that were carbapenem-resistant or 'susceptible, increased exposure' from Greek hospitals that participated in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE, 2013-2014), in the CCRE survey (2019) and in a national follow-up survey (2022) including, for the latter, an estimation of transmission events.ResultsFive K. pneumoniae STs including ST258/512 (n = 101 isolates), ST11 (n = 93), ST39 (n = 56), ST147 (n = 21) and ST323 (n = 13) accounted for more than 90% of CPKP isolates in the dataset. While ST11, ST147 and ST258/512 have been detected in participating hospitals since 2013 and 2014, KPC-2-producing ST39 and ST323 emerged in 2019 and 2022, respectively. Based on the defined genetic relatedness cut-off, 44 within-hospital transmission events were identified in the 2022 survey dataset, with 12 of 15 participating hospitals having at least one within-hospital transmission event.ConclusionThe recent emergence and rapid spread of new high-risk K. pneumoniae clones in the Greek healthcare system related to within-hospital transmission is of concern and highlights the need for molecular surveillance and enhanced infection prevention and control measures.

Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Blockchain-Based Traceability Architecture for Mapping Object-Related Supply Chain Events.

Supply chains have evolved into dynamic, interconnected supply networks, which increases the complexity of achieving end-to-end traceability of object flows and their experienced events. With its capability of ensuring a secure, transparent, and immutable environment without relying on a trusted third party, the emerging blockchain technology shows strong potential to enable end-to-end traceability in such complex multitiered supply networks. This paper aims to overcome the limitations of existing blockchain-based traceability architectures regarding their object-related event mapping ability, which involves mapping the creation and deletion of objects, their aggregation and disaggregation, transformation, and transaction, in one holistic architecture. Therefore, this paper proposes a novel 'blueprint-based' token concept, which allows clients to group tokens into different types, where tokens of the same type are non-fungible. Furthermore, blueprints can include minting conditions, which, for example, are necessary when mapping assembly processes. In addition, the token concept contains logic for reflecting all conducted object-related events in an integrated token history. Finally, for validation purposes, this article implements the architecture's components in code and proves its applicability based on the Ethereum blockchain. As a result, the proposed blockchain-based traceability architecture covers all object-related supply chain events and proves its general-purpose end-to-end traceability capabilities of object flows.

PulseNet International Survey on the Implementation of Whole Genome Sequencing in Low and Middle-Income Countries for Foodborne Disease Surveillance.

PulseNet International (PNI) is a global network of 88 countries who work together through their regional and national public health laboratories to track foodborne disease around the world. The vision of PNI is to implement globally standardized surveillance using whole genome sequencing (WGS) for real-time identification and subtyping of foodborne pathogens to strengthen preparedness and response and lower the burden of disease. Several countries in North America and Europe have experienced significant benefits in disease mitigation after implementing WGS. To broaden the routine use of WGS around the world, challenges and barriers must be overcome. We conducted this study to determine the challenges and barriers countries are encountering in their attempts to implement WGS and to identify how PNI can provide support to improve and become a better integrated system overall. A survey was designed with a set of qualitative questions to capture the status, challenges, barriers, and successes of countries in the implementation of WGS and was administered to laboratories in Africa, Asia-Pacific, Latin America and the Caribbean, and Middle East. One-third of respondents do not use WGS, and only 8% reported using WGS for routine, real-time surveillance. The main barriers for implementation of WGS were lack of funding, gaps in expertise, and training, especially for data analysis and interpretation. Features of an ideal system to facilitate implementation and global surveillance were identified as an all-in-one software that is free, accessible, standardized and validated. This survey highlights the minimal use of WGS for foodborne disease surveillance outside the United States, Canada, and Europe to date. Although funding remains a major barrier to WGS-based surveillance, critical gaps in expertise and availability of tools must be overcome. Opportunities to seek sustainable funding, provide training, and identify solutions for a globally standardized surveillance platform will accelerate implementation of WGS worldwide.

Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls.

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

Quantitative analysis of drug effects at the whole-body level: a case study for glucose metabolism in malaria patients.

We propose a hierarchical modelling approach to construct models for disease states at the whole-body level. Such models can simulate effects of drug-induced inhibition of reaction steps on the whole-body physiology. We illustrate the approach for glucose metabolism in malaria patients, by merging two detailed kinetic models for glucose metabolism in the parasite Plasmodium falciparum and the human red blood cell with a coarse-grained model for whole-body glucose metabolism. In addition we use a genome-scale metabolic model for the parasite to predict amino acid production profiles by the malaria parasite that can be used as a complex biomarker.

Incorporating covalent and allosteric effects into rate equations: the case of muscle glycogen synthase.

Several enzymes have been described that undergo both allosteric and covalent regulation, but, to date, there exists no succinct kinetic description that is able to account for both of these mechanisms of regulation. Muscle glycogen synthase, an enzyme implicated in the pathogenesis of several metabolic diseases, is activated by glucose 6-phosphate and inhibited by ATP and phosphorylation at multiple sites. A kinetic description of glycogen synthase could provide insight into the relative importance of these modifiers. In the present study we show, using non-linear parameter optimization with robust weight estimation, that a Monod-Wyman-Changeux model in which phosphorylation favours the inactive T conformation provides a satisfactory description of muscle glycogen synthase kinetics. The best-fit model suggests that glucose 6-phosphate and ATP compete for the same allosteric site, but that ATP also competes with the substrate UDP-glucose for the active site. The novelty of our approach lies in treating covalent modification as equivalent to allosteric modification. Using the obtained rate equation, the relationship between enzyme activity and phosphorylation state is explored and shown to agree with experimental results. The methodology we propose could also be applied to other enzymes that undergo both allosteric and covalent modification.

Comparison of triploid and diploid rainbow trout (Oncorhynchus mykiss) fine-scale movement, migration and catchability in lowland lakes of western Washington.

Fisheries managers stock triploid (i.e., infertile, artificially produced) rainbow trout Oncorhynchus mykiss in North American lakes to support sport fisheries while minimizing the risk of genetic introgression between hatchery and wild trout. In Washington State, the Washington Department of Fish and Wildlife (WDFW) allocates approximately US $3 million annually to stock hatchery-origin rainbow trout in > 600 lakes, yet only about 10% of them are triploids. Many lakes in Washington State drain into waters that support wild anadromous steelhead O. mykiss that are listed as threatened under the U.S. Endangered Species Act. As a result, there is a strong interest in understanding the costs and benefits associated with stocking sterile, triploid rainbow trout as an alternative to traditional diploids. The objectives of this study were to compare triploid and diploid rainbow trout in terms of: (1) contribution to the sport fishery catch, (2) fine-scale movements within the study lakes, (3) rate of emigration from the lake, and (4) natural mortality. Our results demonstrated that triploid and diploid trout had similar day-night distribution patterns, but triploid trout exhibited a lower emigration rate from the lake and lower catch rates in some lakes. Overall, triploid rainbow trout represent a viable alternative to stocking of diploids, especially in lakes draining to rivers, because they are sterile, have comparable home ranges, and less often migrate.

Integrating genome-based informatics to modernize global disease monitoring, information sharing, and response.

The rapid advancement of genome technologies holds great promise for improving the quality and speed of clinical and public health laboratory investigations and for decreasing their cost. The latest generation of genome DNA sequencers can provide highly detailed and robust information on disease-causing microbes, and in the near future these technologies will be suitable for routine use in national, regional, and global public health laboratories. With additional improvements in instrumentation, these next- or third-generation sequencers are likely to replace conventional culture-based and molecular typing methods to provide point-of-care clinical diagnosis and other essential information for quicker and better treatment of patients. Provided there is free-sharing of information by all clinical and public health laboratories, these genomic tools could spawn a global system of linked databases of pathogen genomes that would ensure more efficient detection, prevention, and control of endemic, emerging, and other infectious disease outbreaks worldwide.

Sensitivity to biases of case-control studies on medical procedures, particularly surgery and blood transfusion, and risk of Creutzfeldt-Jakob disease.

BACKGROUND: Evidence of risk of Creutzfeldt-Jakob disease (CJD) associated with medical procedures, including surgery and blood transfusion, is limited by susceptibility to bias in epidemiological studies. METHODS: Sensitivity to bias was explored using a central-birth-cohort model using data from 18 case-control studies obtained after a review of 494 reports on medical procedures and risk of CJD, systematic for the period January 1, 1989 to December 31, 2011. RESULTS: The validity of the findings in these studies may have been undermined by: recall; control selection; exposure assessment in life-time periods of different duration, out of time-at-risk of effect, or asymmetry in case/control data; and confounding by concomitant blood transfusion at the time of surgery. For sporadic CJD (sCJD), a history of surgery or blood transfusion was associated with risk in some, but not all, recent studies at a ≥10 year lag time, when controls were longitudinally sampled. Space-time aggregation of surgical events was not seen. Surgery at early clinical onset might be overrepresented among cases. Neither surgical history nor blood transfusion unlabelled for donor status, dental treatments or endoscopic examinations were linked to variant CJD (vCJD). CONCLUSIONS: These results indicate the need for further research. Common challenges within these studies include access to and content of past medical/dental treatment records for diseases with long incubation periods.

SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes.

The surfaces of the infected erythrocyte (IE) and the merozoite, two developmental stages of malaria parasites, expose antigenic determinants to the host immune system. We report on surface-associated interspersed genes (surf genes), which encode a novel polymorphic protein family, SURFINs, present on both IEs and merozoites. A SURFIN expressed in 3D7 parasites, SURFIN4.2, was identified by mass spectrometric analysis of peptides cleaved off the surface of live IEs with trypsin. SURFINs are encoded by a family of 10 surf genes, including three predicted pseudogenes, located within or close to the subtelomeres of five of the chromosomes. SURFINs show structural and sequence similarities with exported surface-exposed proteins (PvSTP1, PkSICAvar, PvVIR, Pf332, and PfEMP1) of several Plasmodium species. SURFIN4.2 of a parasite other than 3D7 (FCR3S1.2) showed polymorphisms in the extracellular domain, suggesting sequence variability between genotypes. SURFIN4.2 not only was found cotransported with PfEMP1 and RIFIN to the IE surface, but also accumulated in the parasitophorous vacuole. In released merozoites, SURFIN4.2 was present in an amorphous cap at the parasite apex, where it may be involved in the invasion of erythrocytes. By exposing shared polymorphic antigens on IEs and merozoites, the parasite may coordinate the antigenic composition of these attachment surfaces during growth in the bloodstream.

Draft genome sequencing of giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species?

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16x coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.

Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens.

In this study we describe a novel protocol for rapid molecular analysis of patient samples using a combination of real-time polymerase chain reaction (PCR) and Sanger sequencing. This would normally take 2 working days in the diagnostic laboratory, but using this protocol the process can be completed within 3 h using equipment normally found in the laboratory. The innovative steps in this protocol are the sequencing of the product generated in the diagnostic real-time PCR, addition of a sequencing tail to the PCR primer, which increases the quality of the sequence without loss of sensitivity or specificity, and optimization of the hands-on and instrument steps using modern reagents. The read length of the sequencing step is routinely 250 nucleotides, which is substantially longer than existing rapid sequencing methods, increasing the chances of covering several genetic markers within 1 analysis. As proof of the concept, we used the detection and genotyping of the intestinal parasite Giardia lamblia, but the protocol can be applied to any PCR and sequence-based analysis.

Contrasting landscape influences on sediment supply and stream restoration priorities in northern Fennoscandia (Sweden and Finland) and coastal British Columbia.

Sediment size and supply exert a dominant control on channel structure. We review the role of sediment supply in channel structure, and how regional differences in sediment supply and land use affect stream restoration priorities. We show how stream restoration goals are best understood within a common fluvial geomorphology framework defined by sediment supply, storage, and transport. Land-use impacts in geologically young landscapes with high sediment yields (e.g., coastal British Columbia) typically result in loss of in-stream wood and accelerated sediment inputs from bank erosion, logging roads, hillslopes and gullies. In contrast, northern Sweden and Finland are landscapes with naturally low sediment yields caused by low relief, resistant bedrock, and abundant mainstem lakes that act as sediment traps. Land-use impacts involved extensive channel narrowing, removal of obstructions, and bank armouring with boulders to facilitate timber floating, thereby reducing sediment supply from bank erosion while increasing export through higher channel velocities. These contrasting land-use impacts have pushed stream channels in opposite directions (aggradation versus degradation) within a phase-space defined by sediment transport and supply. Restoration in coastal British Columbia has focused on reducing sediment supply (through bank and hillslope stabilization) and restoring wood inputs. In contrast, restoration in northern Fennoscandia (Sweden and Finland) has focused on channel widening and removal of bank-armouring boulders to increase sediment supply and retention. These contrasting restoration priorities illustrate the consequences of divergent regional land-use impacts on sediment supply, and the utility of planning restoration activities within a mechanistic sediment supply-transport framework.

Discovery and application of insertion-deletion (INDEL) polymorphisms for QTL mapping of early life-history traits in Atlantic salmon.

BACKGROUND: For decades, linkage mapping has been one of the most powerful and widely used approaches for elucidating the genetic architecture of phenotypic traits of medical, agricultural and evolutionary importance. However, successful mapping of Mendelian and quantitative phenotypic traits depends critically on the availability of fast and preferably high-throughput genotyping platforms. Several array-based single nucleotide polymorphism (SNP) genotyping platforms have been developed for genetic model organisms during recent years but most of these methods become prohibitively expensive for screening large numbers of individuals. Therefore, inexpensive, simple and flexible genotyping solutions that enable rapid screening of intermediate numbers of loci (approximately 75-300) in hundreds to thousands of individuals are still needed for QTL mapping applications in a broad range of organisms. RESULTS: Here we describe the discovery of and application of insertion-deletion (INDEL) polymorphisms for cost-efficient medium throughput genotyping that enables analysis of >75 loci in a single automated sequencer electrophoresis column with standard laboratory equipment. Genotyping of INDELs requires low start-up costs, includes few standard sample handling steps and is applicable to a broad range of species for which expressed sequence tag (EST) collections are available. As a proof of principle, we generated a partial INDEL linkage map in Atlantic salmon (Salmo salar) and rapidly identified a number of quantitative trait loci (QTLs) affecting early life-history traits that are expected to have important fitness consequences in the natural environment. CONCLUSIONS: The INDEL genotyping enabled fast coarse-mapping of chromosomal regions containing QTL, thus providing an efficient means for characterization of genetic architecture in multiple crosses and large pedigrees. This enables not only the discovery of larger number of QTLs with relatively smaller phenotypic effect but also provides a cost-effective means for evaluation of the frequency of segregating QTLs in outbred populations which is important for further understanding how genetic variation underlying phenotypic traits is maintained in the wild.

Comparison of the AF146527 and B1 repeated elements, two real-time PCR targets used for detection of Toxoplasma gondii.

Previous studies have reported the increased sensitivity of PCR targeting AF146527 over that of PCR targeting the B1 gene for diagnosis of toxoplasmosis. The present study suggests that the AF146527 element was absent in 4.8% of human Toxoplasma gondii-positive samples tested. The data argue that the B1 gene may be the preferred diagnostic target.

The cancer's nervous tooth: Considering the neuronal crosstalk within tumors.

The nervous system is a superordinate organ in the body that controls the function of virtually all other organs and tissues. In the past, the role of the nervous system in cancer development and progression has largely been ascribed to an immunosuppressive function, which saps the immune system's ability to respond to a tumor. However, it is now clear that direct interactions of tumor cells with nerve cells occur, too. We herein provide arguments for the hypothesis that tumors initiate their own innervation by the release of neurotrophic factors including the nerve growth factor, the brain-derived growth factor, and the vascular endothelial growth factor. By this process, which we have termed neoneurogenesis, the tumor cells get in close contact to the nerve cells, forming a neuro-neoplastic synapse. Through these synapses, neurotransmitters are directly supplied to the tumors, which has impact on tumor growth and metastasis formation.

Concordance of SNP- and allele-based typing workflows in the context of a large-scale international Salmonella Enteritidis outbreak investigation.

A large European multi-country Salmonella enterica serovar Enteritidis outbreak associated with Polish eggs was characterized by whole-genome sequencing (WGS)-based analysis, with various European institutes using different analysis workflows to identify isolates potentially related to the outbreak. The objective of our study was to compare the output of six of these different typing workflows (distance matrices of either SNP-based or allele-based workflows) in terms of cluster detection and concordance. To this end, we analysed a set of 180 isolates coming from confirmed and probable outbreak cases, which were representative of the genetic variation within the outbreak, supplemented with 22 unrelated contemporaneous S. enterica serovar Enteritidis isolates. Since the definition of a cluster cut-off based on genetic distance requires prior knowledge on the evolutionary processes that govern the bacterial populations in question, we used a variety of hierarchical clustering methods (single, average and complete) and selected the optimal number of clusters based on the consensus of the silhouette, Dunn2, and McClain-Rao internal validation indices. External validation was done by calculating the concordance with the WGS-based case definition (SNP-address) for this outbreak using the Fowlkes-Mallows index. Our analysis indicates that with complete-linkage hierarchical clustering combined with the optimal number of clusters, as defined by three internal validity indices, the six different allele- and SNP-based typing workflows generate clusters with similar compositions. Furthermore, we show that even in the absence of coordinated typing procedures, but by using an unsupervised machine learning methodology for cluster delineation, the various workflows that are currently in use by six European public-health authorities can identify concordant clusters of genetically related S. enterica serovar Enteritidis isolates; thus, providing public-health researchers with comparable tools for detection of infectious-disease outbreaks.