Search

Plasmids found in Acinetobacter species contribute to the spread of antibiotic resistance genes. They appear to be largely confined to this genus and cannot be typed with available tools and databases. Here, a method for distinguishing and typing these plasmids was developed using a curated, non-redundant set of 621 complete sequences of plasmids from Acinetobacter baumannii. Plasmids were separated into 3 groups based on the Pfam domains of the encoded replication initiation (Rep) protein and a fourth group that lack an identifiable Rep protein. The

Oxford Nanopore Technologies (ONT) sequencing has rich potential for genomic epidemiology and public health investigations of bacterial pathogens, particularly in low-resource settings and at the point of care, due to its portability and affordability. However, low base-call accuracy has limited the reliability of ONT data for critical tasks such as antimicrobial resistance (AMR) and virulence gene detection and typing, serotype prediction, and cluster identification. Thus, Illumina sequencing remains the standard for genomic surveillance despite higher capital and running costs. We tested the accuracy of ONT-only assemblies for common applied bacterial genomics tasks (genotyping and cluster detection, implemented via Kleborate, Kaptive and Pathogenwatch), using data from 54 unique Klebsiella pneumoniae isolates. ONT reads generated via MinION with R9.4.1 flowcells were basecalled using three alternative models [Fast, High-accuracy (HAC) and Super-accuracy (SUP), available within ONT’s Guppy software], assembled with Flye and polished using Medaka. Accuracy of typing using ONT-only assemblies was compared with that of Illumina-only and hybrid ONT+Illumina assemblies, constructed from the same isolates as reference standards. The most resource-intensive ONT-assembly approach (SUP basecalling, with or without Medaka polishing) performed best, yielding reliable capsule (K) type calls for all strains (100 % exact or best matching locus), reliable multi-locus sequence type (MLST) assignment (98.3 % exact match or single-locus variants), and good detection of acquired AMR genes and mutations (88–100 % correct identification across the various drug classes). Distance-based trees generated from SUP+Medaka assemblies accurately reflected overall genetic relationships between isolates. The definition of outbreak clusters from ONT-only assemblies was problematic due to inflation of SNP counts by high base-call errors. However, ONT data could be reliably used to ‘rule out’ isolates of distinct lineages from suspected transmission clusters. HAC basecalling + Medaka polishing performed similarly to SUP basecalling without polishing. Therefore, we recommend investing compute resources into basecalling (SUP model), wherever compute resources and time allow, and note that polishing is also worthwhile for improved performance. Overall, our results show that MLST, K type and AMR determinants can be reliably identified with ONT-only R9.4.1 flowcell data. However, cluster detection remains challenging with this technology.

The outer polysaccharide capsule and lipopolysaccharide (LPS) antigens are key targets for novel control strategies targeting

Background Peer comparison audit and feedback has demonstrated effectiveness in improving antibiotic prescribing practices, but only a minority of prescribers view their reports. We rigorously tested 3 behavioral nudging techniques delivered by email to improve report opening. Methods We conducted a pragmatic randomized controlled trial among Ontario long-term care prescribers enrolled in an ongoing peer comparison audit and feedback program which includes data on their antibiotic prescribing patterns. Physicians were randomized to 1 of 8 possible sequences of intervention/control allocation to 3 different behavioral email nudges: a social peer comparison nudge (January 2020), a maintenance of professional certification incentive nudge (October 2020), and a prior participation nudge (January 2021). The primary outcome was feedback report opening; the primary analysis pooled the effects of all 3 nudging interventions. Results The trial included 421 physicians caring for >28 000 residents at 450 facilities. In the pooled analysis, physicians opened only 29.6% of intervention and 23.9% of control reports (odds ratio [OR], 1.51 [95% confidence interval {CI}, 1.10–2.07], P = .011); this difference remained significant after accounting for physician characteristics and clustering (adjusted OR [aOR], 1.74 [95% CI, 1.24–2.45], P = .0014). Of individual nudging techniques, the prior participation nudge was associated with a significant increase in report opening (OR, 1.62 [95% CI, 1.06–2.47], P = .026; aOR, 2.16 [95% CI, 1.33–3.50], P = .0018). In the pooled analysis, nudges were also associated with accessing more report pages (aOR, 1.28 [95% CI, 1.14–1.43], P Conclusions Enhanced nudging strategies modestly improved report opening, but more work is needed to optimize physician engagement with audit and feedback. Clinical Trials Registration NCT04187742.

Bed management is one of the most crucial tasks in a hospital. A desktop-based (PC) information technology system has been widely adopted to assist this operation. The fixed location and the limited amount of available PCs shared among staff for different routine tasks can cause delays in bed information updating and therefore delays to the bed management processes. With the penetration of smartphones, a mobile-based application becomes a reliable alternative solution for bed management to complement the existing system. Smartphones offer a few benefits, especially mobility because users can update the information from any place and at any time. Our aim was to study the applicability of a mobile-based application for assisting bed management operation. The application consisted of ward-based bed information visualization, bed information updating, and ward information updating modules. Observation and interviews with expert users were performed to outline the required functions of the application. On a Likert 1-5 scale, the mobile application achieved a mean score of 3.45 for usefulness, 3.40 for ease of use, and 3.42 for satisfaction. Meanwhile, the respondents were also concerned about the mobile data consumption of the app, which took up 45.8% of the negative feedback. On the positive side, the respondents agree that the mobile application helps them in the bed management operation.

Klebsiella pneumoniae is a common cause of antimicrobial-resistant opportunistic infections in hospitalized patients. The species is naturally resistant to penicillins, and members of the population often carry acquired resistance to multiple antimicrobials. However, knowledge of K. pneumoniae ecology, population structure or pathogenicity is relatively limited. Over the past decade, K. pneumoniae has emerged as a major clinical and public health threat owing to increasing prevalence of healthcare-associated infections caused by multidrug-resistant strains producing extended-spectrum β-lactamases and/or carbapenemases. A parallel phenomenon of severe community-acquired infections caused by 'hypervirulent' K. pneumoniae has also emerged, associated with strains expressing acquired virulence factors. These distinct clinical concerns have stimulated renewed interest in K. pneumoniae research and particularly the application of genomics. In this Review, we discuss how genomics approaches have advanced our understanding of K. pneumoniae taxonomy, ecology and evolution as well as the diversity and distribution of clinically relevant determinants of pathogenicity and antimicrobial resistance. A deeper understanding of K. pneumoniae population structure and diversity will be important for the proper design and interpretation of experimental studies, for interpreting clinical and public health surveillance data and for the design and implementation of novel control strategies against this important pathogen.

Klebsiella pneumoniaeis a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study ofK. pneumoniaeclinical isolates identified in a hospital microbiological diagnostic laboratory. Disease burden was two-thirds urinary tract infections (UTI; associated with female sex and age), followed by pneumonia (15%), wound (10%) and disseminated infections/sepsis (10%). Whole-genome sequencing (WGS) revealed a diverse pathogen population, including other species within theK. pneumoniaecomplex (18%). Several infections were caused byK. variicola/K. pneumoniaespecies hybrids, one of which showed evidence of nosocomial transmission, indicating fitness to transmit and cause disease despite a lack of acquired antimicrobial resistance (AMR). A wide range of AMR phenotypes were observed and, in most cases, corresponding mechanisms were identified in the genomes, mainly in the form of plasmid-borne genes. ESBLs were correlated with presence of other acquired AMR genes (median 10). Bacterial genomic features associated with nosocomial onset of disease were ESBL genes (OR 2.34, p=0.015) and rhamnose-positive capsules (OR 3.12, p-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate the underlying burden ofK. pneumoniaedisease in hospitalised patients is due largely to opportunistic infections with diverse strains. However, we also identified several successful lineages that were overrepresented but not due to nosocomial transmission. These lineages were associated with ESBL, yersiniabactin, mannose+ K loci and rhamnose- K loci; most are also common in public clinical genome collections, suggesting enhanced propensity for colonisation and spread in the human population.

The outer polysaccharide capsule and lipopolysaccharide antigens are key targets for novel control strategies targeting Klebsiella pneumoniae and related taxa from the K. pneumoniae species complex (KpSC), including vaccines, phage and monoclonal antibody therapies. Given the importance and growing interest in these highly diverse surface antigens, we had previously developed Kaptive, a tool for rapidly identifying and typing capsule (K) and outer lipopolysaccharide (O) loci from whole genome sequence data. Here, we report two significant updates, now freely available in Kaptive 2.0 (github.com/katholt/kaptive); i) the addition of 16 novel K locus sequences to the K locus reference database following an extensive search of >17,000 KpSC genomes; and ii) enhanced O locus typing to enable prediction of the clinically relevant O2 antigen (sub)types, for which the genetic determinants have been recently described. We applied Kaptive 2.0 to a curated dataset of >12,000 public KpSC genomes to explore for the first time the distribution of predicted O (sub)types across species, sampling niches and clones, which highlighted key differences in the distributions that warrant further investigation. As the uptake of genomic surveillance approaches continues to expand globally, the application of Kaptive 2.0 will generate novel insights essential for the design of effective KpSC control strategies.Significance as a BioResource to the communityKlebsiella pneumoniae is a major cause of bacterial healthcare associated infections globally, with increasing rates of antimicrobial resistance, including strains with resistance to the drugs of last resort. The latter have therefore been flagged as priority pathogens for the development of novel control strategies.K. pneumoniae produce two key surface antigen sugars (capsular polysaccharide and lipopolysaccharide (LPS)) that are immunogenic and targets for novel controls such as a vaccines and phage therapy. However, there is substantial antigenic diversity in the population and relatively little is understood about the distribution of antigen types geographically and among strains causing different types of infections. Whereas laboratory-based antigen typing is difficult and rarely performed, information about the relevant synthesis loci can be readily extracted from whole genome sequence data. We have previously developed Kaptive, a freely available tool for rapid typing of Klebsiella capsule and LPS loci from genome sequences.Kaptive is now used widely in the global research community and has facilitated new insights into Klebsiella capsule and LPS diversity. Here we present an update to Kaptive facilitating i) the identification of 16 additional novel capsule loci, and ii) the prediction of immunologically relevant LPS O2 antigen subtypes. These updates will enable enhanced sero-epidemiological surveillance for K. pneumoniae, to inform the design of vaccines and other novel Klebsiella control strategies.Data summaryThe updated code and reference databases for Kaptive are available at https://github.com/katholt/KaptiveGenome accessions from which reference sequences of novel K loci were defined are listed in Supplementary Table 1, and genomes from which these loci were detected (along with the corresponding Kaptive output) are listed in Supplementary Table 2.Accessions for the genomes screened for O types/subtypes (along with the corresponding Kaptive output) are listed in Supplementary Table 3.The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.Repositories1.1RepositoriesGenome sequence from which the novel K locus KL182 was defined has been deposited under the accession JAJHNT000000000.

Klebsiella pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes., Klebsiella pneumoniae is a pathogen of increasing public health concern and antimicrobial resistance is becoming more prevalent. Here, the authors describe a K. pneumoniae genotyping tool, Kleborate, that can be used to identify lineages and detect antimicrobial resistance and virulence loci.

Antimicrobial agents have been used in meat production for decades and its consumption is considered an key driver for the emergence and dissemination of antimicrobial resistance (AMR). However, large-scale studies on AMR changes in animal isolates since the introduction of antimicrobial usage remain scarce. We applied whole genome sequencing analysis to 982 animal-derived Escherichia coli collected in China from 1970s to 2019 and found increasing trends for the presence of numerous antimicrobial resistance genes (ARGs), including those conferring resistance to critically important agents for veterinary (florfenicol and norfloxacin) and human medicine (colistin, cephalosporins, and meropenem). Extensive diversity and increasing complexity of ARGs and their associated mobile genetic elements (MGEs) such as plasmids were also observed. The plasmids, IncC, IncHI2, IncK, IncI, IncX and IncF played a key role as highly effective vehicles for disseminating ARGs. Correlation analysis also revealed an association between antimicrobial production and emergence of ARGs at a spatial and temporal level. Prohibiting or strictly curtailing antimicrobial use in animals will potentially negate the current trends of AMR as the bacterial genome is highly changeable and using different drugs of the same class, or even unrelated classes, may co-select for MGEs carrying a plethora of co-existing ARGs. Therefore, limiting or ceasing antimicrobial use in animals to control AMR requires careful consideration.

We present the photometric analysis of Gaia19bld, a high-magnification ($A\approx60$) microlensing event located in the southern Galactic plane, which exhibited finite source and microlensing parallax effects. Due to a prompt detection by the Gaia satellite and the very high brightness of $I = 9.05~$mag at the peak, it was possible to collect a complete and unique set of multi-channel follow-up observations, which allowed us to determine all parameters vital for the characterisation of the lens and the source in the microlensing event. Gaia19bld was discovered by the Gaia satellite and was subsequently intensively followed up with a network of ground-based observatories and the Spitzer Space Telescope. We collected multiple high-resolution spectra with Very Large Telescope (VLT)/X-Shooter to characterise the source star. The event was also observed with VLT Interferometer (VLTI)/PIONIER during the peak. Here we focus on the photometric observations and model the light curve composed of data from Gaia, Spitzer, and multiple optical, ground-based observatories. We find the best-fitting solution with parallax and finite source effects. We derived the limit on the luminosity of the lens based on the blended light model and spectroscopic distance. We compute the mass of the lens to be $1.13 \pm 0.03~M_{\odot}$ and derive its distance to be $5.52^{+0.35}_{-0.64}~\mathrm{kpc}$. The lens is likely a main sequence star, however its true nature has yet to be verified by future high-resolution observations. Our results are consistent with interferometric measurements of the angular Einstein radius, emphasising that interferometry can be a new channel for determining the masses of objects that would otherwise remain undetectable, including stellar-mass black holes., Comment: accepted to Astronomy&Astrophysics

K. pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes.

The evolution of traits including antibiotic resistance, virulence, and increased fitness in Klebsiella pneumoniae and related species has been linked to the acquisition of mobile genetic elements through horizontal transfer. Among them, genomic islands (GIs) preferentially integrating at genes encoding tRNAs and the tmRNA (t(m)DNAs) would be significant in promoting chromosomal diversity. Here, we studied the whole set of t(m)DNAs present in 66 Klebsiella chromosomes, investigating their usage as integration sites and the properties of the integrated GIs. A total of 5,624 t(m)DNAs were classified based on their sequence conservation, genomic context, and prevalence. 161 different GIs and prophages were found at these sites, hosting 3,540 gene families including various related to virulence and drug resistance. Phylogenetic analyses supported the acquisition of several of these elements through horizontal gene transfer, likely mediated by a highly diverse set of encoded integrases targeting specific t(m)DNAs and sublocations inside them. Only a subset of the t(m)DNAs had integrated GIs and even identical tDNA copies showed dissimilar usage frequencies, suggesting that the genomic context would influence the integration site selection. This usage bias, likely towards avoiding disruption of polycistronic transcriptional units, would be conserved across Gammaproteobacteria. The systematic comparison of the t(m)DNAs across different strains allowed us to discover an unprecedented number of K. pneumoniae GIs and prophages and to raise important questions and clues regarding the fundamental properties of t(m)DNAs as targets for the integration of mobile genetic elements and drivers of bacterial genome evolution and pathogen emergence.

Background Klebsiella pneumoniae is a leading cause of bloodstream infection (BSI). Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial-resistant (AMR) K. pneumoniae and also for the characteristically antimicrobial-sensitive, community-acquired “hypervirulent” strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. Methods We conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate, a K. pneumoniae-specific genomic typing tool. Results K. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common among South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes among K. pneumoniae. Conclusions K. pneumoniae BSI in South and Southeast Asia are caused by different STs from those predominating in other regions, and with higher frequency of acquired virulence determinants. K. pneumoniae carrying both iuc and AMR genes were also detected at higher rates than have been reported elsewhere. The study demonstrates how genomics-based surveillance—reporting full molecular profiles including STs, AMR, virulence and serotype locus information—can help standardise comparisons between sites and identify regional differences in pathogen populations.

Klebsiella pneumoniae is a common cause of antimicrobial-resistant opportunistic infections in hospitalized patients. The species is naturally resistant to penicillins, and members of the population often carry acquired resistance to multiple antimicrobials. However, knowledge of K. pneumoniae ecology, population structure or pathogenicity is relatively limited. Over the past decade, K. pneumoniae has emerged as a major clinical and public health threat owing to increasing prevalence of healthcare-associated infections caused by multidrug-resistant strains producing extended-spectrum β-lactamases and/or carbapenemases. A parallel phenomenon of severe community-acquired infections caused by 'hypervirulent' K. pneumoniae has also emerged, associated with strains expressing acquired virulence factors. These distinct clinical concerns have stimulated renewed interest in K. pneumoniae research and particularly the application of genomics. In this Review, we discuss how genomics approaches have advanced our understanding of K. pneumoniae taxonomy, ecology and evolution as well as the diversity and distribution of clinically relevant determinants of pathogenicity and antimicrobial resistance. A deeper understanding of K. pneumoniae population structure and diversity will be important for the proper design and interpretation of experimental studies, for interpreting clinical and public health surveillance data and for the design and implementation of novel control strategies against this important pathogen.

Severe liver abscess infections caused by hypervirulent clonal-group CG23 Klebsiella pneumoniae have been increasingly reported since the mid-1980s. Strains typically possess several virulence factors including an integrative, conjugative element ICEKp encoding the siderophore yersiniabactin and genotoxin colibactin. Here we investigate CG23’s evolutionary history, showing several deep-branching sublineages associated with distinct ICEKp acquisitions. Over 80% of liver abscess isolates belong to sublineage CG23-I, which emerged in ~1928 following acquisition of ICEKp10 (encoding yersiniabactin and colibactin), and then disseminated globally within the human population. CG23-I’s distinguishing feature is the colibactin synthesis locus, which reportedly promotes gut colonisation and metastatic infection in murine models. These data show circulation of CG23 K. pneumoniae decades before the liver abscess epidemic was first recognised, and provide a framework for future epidemiological and experimental studies of hypervirulent K. pneumoniae. To support such studies we present an open access, completely sequenced CG23-I human liver abscess isolate, SGH10., Since the 1980s, hypervirulent clonal-group CG23 serotype K1 Klebsiella pneumoniae has been recognised as a prominent cause of community-acquired liver abscess and other severe infections. Here, the authors investigate the genomic evolutionary history of CG23 and suggest a new reference strain for CG23.

Klebsiella pneumoniae (Kp) is an infamous cause of multi-drug resistant (MDR) healthcare-associated infections and several MDR clones are globally distributed. A small number of drug-susceptible clones have also become globally distributed, causing severe community-acquired infections. These ‘hypervirulent’ clones are distinguished by expression of highly serum-resistant K1/K2 capsules, plus high prevalence of acquired virulence determinants. While hypervirulence and drug resistance are usually mutually exclusive, there are now increasing reports of convergent strains that are both highly virulent and MDR – a potentially disastrous combination. To better understand the risks of MDR-virulence convergence, we leveraged a collection of >2200 Kp genomes to identify 28 common clones (n≥10 genomes each), and performed a genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified by acquired resistance and virulence gene prevalence. Chromosomal recombination, capsule locus diversity, pan-genome, plasmid and phage dynamics were compared. MDR clones were highly diverse, with frequent chromosomal recombination generating extensive capsule locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which were each associated with only a single capsule locus and showed significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. These data suggest that hypervirulent clones are subject to some sort of constraint for horizontal gene-transfer. Hence we predict MDR clones pose the greatest risk for MDR-virulence convergence because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

A retrospective genomic study led to the identification of two carbapenem-resistant K. pneumoniae isolates (KPN535 and KPC45) carrying bla KPC-2 genes on non-conjugative plasmids.….

Background: MDR and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options. Objectives: Our aim was to characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15. Methods: The complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing. Results: Both isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and seven other AMR genes (blaTEM, aac3'-IIa, dfrA1, satA2, blaSHV, sul1 and aadA1). The other carried remnants of these elements encoding blaTEM and aac3'-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania. Conclusions: The presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance. publishedVersion

Klebsiella pneumoniae has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and drug susceptible community-acquired invasive infections. These pathotypes are generally associated with two distinct subsets of K. pneumoniae lineages or ‘clones’ that are distinguished by the presence of acquired resistance genes and several key virulence loci. Genomic evolutionary analyses of the most notorious MDR and invasive community-associated (‘hypervirulent’) clones indicate differences in terms of chromosomal recombination dynamics and capsule polysaccharide diversity, but it remains unclear if these differences represent generalised trends. Here we leverage a collection of >2200 K. pneumoniae genomes to identify 28 common clones (n ≥ 10 genomes each), and perform the first genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified on the basis of acquired resistance and virulence gene prevalence. Chromosomal recombination, surface polysaccharide locus diversity, pan-genome, plasmid and phage dynamics were characterised and compared. The data showed that MDR clones were highly diverse, with frequent chromosomal recombination generating extensive surface polysaccharide locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which also showed a significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. Hence the data indicate that hypervirulent clones may be subject to some sort of constraint for horizontal gene transfer that does not apply to the MDR clones. Our findings are relevant for understanding the risk of emergence of individual K. pneumoniae strains carrying both virulence and acquired resistance genes, which have been increasingly reported and cause highly virulent infections that are extremely difficult to treat. Specifically, our data indicate that MDR clones pose the greatest risk, because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes., Author summary Klebsiellla pneumoniae is classified by the World Health Organization as a priority drug-resistant organism because it causes a significant burden of hospital infections that are extremely difficult to treat. However, outside of the hospital setting this bacterium is also an important cause of severe drug-susceptible infections. Until recently these two infection types were associated with distinct subsets of the K. pneumoniae population harbouring high prevalence of drug-resistance and virulence genes, respectively. However, there are now increasing reports of highly-virulent and difficult-to-treat K. pneumoniae strains that carry both resistance and virulence genes. In this study, we used genomic analyses to characterise and compare the evolutionary histories of drug-resistant and virulent K. pneumoniae. We show that the former are highly diverse, frequently acquiring novel genes through the processes of chromosomal recombination, plasmid and bacteriophage acquisition. In comparison, the latter show considerably lower gene content diversity, suggesting that they may be subject to some sort of limitation for gene acquisition. Consequently, we predict that drug-resistant K. pneumoniae are more likely to acquire virulence genes than virulent K. pneumoniae are to acquire drug-resistance genes.

SYNOPSISBackgroundMultidrug resistance (MDR) and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options.ObjectivesTo characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15.MethodsThe complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing.ResultsBoth isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and eight other AMR genes (blaTEM, aac3’-IIa, aph3’-Ia, dfrA1, satA2, blaSHV, sul1, aadA1). The other carried remnants of these elements encoding blaTEM and aac3’-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania.ConclusionsThe presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance.

BackgroundKlebsiella pneumoniaeis a recognised agent of multidrug-resistant (MDR) healthcare-associated infections, however individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin (iuc) and salmochelin (iro) are associated with invasive disease and are common amongst hypervirulentK. pneumoniaeclones that cause severe community-associated infections such as liver abscess and pneumonia. Concerninglyiuchas also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in theK. pneumoniaepopulation.MethodsHere we examined the genetic diversity, distribution and mobilisation ofiucandiroloci among 2503K. pneumoniaegenomes using comparative genomics approaches, and developed tools for tracking them via genomic surveillance.ResultsIroandiucwere detected at low prevalence (iroand sixiuclineages that show distinct patterns of mobilisation and dissemination in theK. pneumoniaepopulation. The major burden ofiucandiroamongst the genomes analysed was due to two linked lineages (iuc1/iro1, 74% andiuc2/iro2, 14%), each carried by a distinct non-self-transmissible IncFIBKvirulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy (rmpA) determinants and include all previously described virulence plasmids ofK. pneumoniae. The otheriucandirolineages were associated with diverse plasmids, including some carrying FII conjugative transfer regions and some imported fromE. coli; the exceptions wereiro3(mobilised byICEKp1), andiuc4(fixed in the chromosome ofK. pneumoniaesubspeciesrhinoscleromatis).Iro/iucMGEs appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc), but were also detected at low prevalence in others such as MDR strain ST258.ConclusionsIucandiroare mobilised inK. pneumoniaevia a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDRK. pneumoniaestrains.

Mobile genetic elements (MGEs) that frequently transfer within and between bacterial species play a critical role in bacterial evolution, and often carry key accessory genes that associate with a bacteria’s ability to cause disease. MGEs carrying antimicrobial resistance (AMR) and/or virulence determinants are common in opportunistic pathogenKlebsiella pneumoniae, which are a leading cause of highly drug-resistant infections in hospitals. Well-characterised virulence determinants inK. pneumoniaeinclude the polyketide synthesis lociybtandclb(also known aspks), encoding the iron-scavenging siderophore yersiniabactin and genotoxin colibactin respectively. These loci are located within an MGE called ICEKp, which is the most common virulence-associated MGE ofK. pneumoniae,providing a mechanism for these virulence factors to spread within the population.Here we apply population genomics to investigate the prevalence, evolution and mobility ofybtandclbinK. pneumoniaepopulations through comparative analysis of 2,498 whole genome sequences. Theybtlocus was detected in 40% ofK. pneumoniaegenomes, particularly amongst those associated with invasive infections. We identified 17 distinctybtlineages and 3clblineages, each associated with one of 14 different structural variants of ICEKp. Comparison with the wider Enterobacteriaceae population showed occasional ICEKpacquisition by other members. Theclblocus was present in 14% of allK. pneumoniaeand 38.4% ofybt+ genomes. Hundreds of independent ICEKpintegration events were detected affecting hundreds of phylogenetically distinctK. pneumoniaelineages, including ≥19 in the globally-disseminated carbapenem-resistant clone CG258. A novel plasmid-encoded form ofybtwas also identified, representing a new mechanism forybtdispersal inK. pneumoniaepopulations. These data show that MGEs carryingybtandclbcirculate freely in theK. pneumoniaepopulation, including among multidrug-resistant strains, and should be considered a target for genomic surveillance along with AMR determinants.AUTHOR SUMMARYKlebsiella pneumoniaeinfections are becoming increasingly difficult to treat with antibiotics. SomeK. pneumoniaestrains also carry extra genes that allow them to synthesise yersiniabactin, an iron-scavenging molecule, which enhances their ability to cause disease. These genes are located on a genetic element that can easily transfer between strains. Here, we screened 2498K. pneumoniaegenome sequences and found substantial diversity in the yersiniabactin genes and the associated genetic elements, including a novel mechanism of transfer, and detected hundreds of distinct yersiniabactin acquisition events betweenK. pneumoniaestrains. We show that these yersiniabactin mobile genetic elements are specifically adapted to theK. pneumoniaepopulation but also occasionally acquired by other bacterial members belonging to the Enterobacteriaceae family such asE. coli.These insights into the movement and genetics of yersiniabactin genes allow tracking of the evolution and spread of yersiniabactin in globalK. pneumoniaepopulations and monitoring for acquisition of yersiniabactin in antibiotic-resistant strains.

Alcohol-based disinfectants and particularly hand rubs are a key way to control hospital infections worldwide. Such disinfectants restrict transmission of pathogens, such as multidrug-resistant Staphylococcus aureus and Enterococcus faecium Despite this success, health care infections caused by E. faecium are increasing. We tested alcohol tolerance of 139 hospital isolates of E. faecium obtained between 1997 and 2015 and found that E. faecium isolates after 2010 were 10-fold more tolerant to killing by alcohol than were older isolates. Using a mouse gut colonization model of E. faecium transmission, we showed that alcohol-tolerant E. faecium resisted standard 70% isopropanol surface disinfection, resulting in greater mouse gut colonization compared to alcohol-sensitive E. faecium We next looked for bacterial genomic signatures of adaptation. Alcohol-tolerant E. faecium accumulated mutations in genes involved in carbohydrate uptake and metabolism. Mutagenesis confirmed the roles of these genes in the tolerance of E. faecium to isopropanol. These findings suggest that bacterial adaptation is complicating infection control recommendations, necessitating additional procedures to prevent E. faecium from spreading in hospital settings.

Mobile genetic elements (MGEs) that frequently transfer within and between bacterial species play a critical role in bacterial evolution, and often carry key accessory genes that associate with a bacteria’s ability to cause disease. MGEs carrying antimicrobial resistance (AMR) and/or virulence determinants are common in the opportunistic pathogen Klebsiella pneumoniae, which is a leading cause of highly drug-resistant infections in hospitals. Well-characterised virulence determinants in K. pneumoniae include the polyketide synthesis loci ybt and clb (also known as pks), encoding the iron-scavenging siderophore yersiniabactin and genotoxin colibactin, respectively. These loci are located within an MGE called ICEKp, which is the most common virulence-associated MGE of K. pneumoniae, providing a mechanism for these virulence factors to spread within the population. Here we apply population genomics to investigate the prevalence, evolution and mobility of ybt and clb in K. pneumoniae populations through comparative analysis of 2498 whole-genome sequences. The ybt locus was detected in 40 % of K. pneumoniae genomes, particularly amongst those associated with invasive infections. We identified 17 distinct ybt lineages and 3 clb lineages, each associated with one of 14 different structural variants of ICEKp. Comparison with the wider population of the family Enterobacteriaceae revealed occasional ICEKp acquisition by other members. The clb locus was present in 14 % of all K. pneumoniae and 38.4 % of ybt+ genomes. Hundreds of independent ICEKp integration events were detected affecting hundreds of phylogenetically distinct K. pneumoniae lineages, including at least 19 in the globally-disseminated carbapenem-resistant clone CG258. A novel plasmid-encoded form of ybt was also identified, representing a new mechanism for ybt dispersal in K. pneumoniae populations. These data indicate that MGEs carrying ybt and clb circulate freely in the K. pneumoniae population, including among multidrug-resistant strains, and should be considered a target for genomic surveillance along with AMR determinants.

Alcohol-based disinfectants and particularly hand rubs are a key way to control hospital infections worldwide. Such disinfectants restrict transmission of pathogens, such as multidrug-resistant

Since the mid-1980s there have been increasing reports of severe community-acquired pyogenic liver abscess, meningitis and bloodstream infections caused by hypervirulentKlebsiella pneumoniae, predominantly encompassing clonal group (CG) 23 serotype K1 strains. Common features of CG23 include a virulence plasmid associated with iron scavenging and hypermucoidy, and a chromosomal integrative and conjugative element (ICE) encoding the siderophore yersiniabactin and the genotoxin colibactin. Here we investigate the evolutionary history and genomic diversity of CG23 based on comparative analysis of 98 genomes. Contrary to previous reports with more limited samples, we show that CG23 comprises several deep branching sublineages dating back to the 1870s, many of which are associated with distinct chromosomal insertions of ICEs encoding yersiniabactin. We find that most liver abscess isolates (>80%) belong to a dominant sublineage, CG23-I, which emerged in the 1920s following acquisition of ICEKp10(encoding colibactin in addition to yersiniabactin) and has undergone clonal expansion and global dissemination within the human population. The unique genomic feature of CG23-I is the production of colibactin, which has been reported previously as a promoter of gut colonisation and dissemination to the liver and brain in a mouse model of CG23K. pneumoniaeinfection, and has been linked to colorectal cancer. We also identify an antibiotic-resistant subclade of CG23-I associated with sexually-transmitted infections in horses dating back to the 1980s. These data show that hypervirulent CG23K. pneumoniaewas circulating in humans for decades before the liver abscess epidemic was first recognised, and has the capacity to acquire and maintain AMR plasmids. These data provide a framework for future epidemiological and experimental studies of hypervirulentK. pneumoniae. To further support such studies we present an open access and completely sequenced human liver abscess isolate, SGH10, which is typical of the globally disseminated CG23-I sublineage.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specialized conditions of hospital and healthcare environments.

Soft tissue sarcomas (STS) are heterogeneous malignant tumors of mesenchymal origin. Due to low incidence and high number of different histological subtypes, their pathogenesis and thus potential targets for their therapy remain barely investigated. Several studies revealed significant higher EPHB4 expression in malignancies such as prostate and colorectal cancer showing survival advantages for these tumor cells. Therefore we studied the expression of EPHB4 in a total of 46 clinical human specimens of different STS and human fibroblasts. EPHB4 mRNA and protein expression were significantly increased in synovial sarcoma. After targeting EPHB4 in fibrosarcoma, synovial sarcoma, liposarcoma and MFH sarcoma cell lines by siRNA or by inhibition of autophosphorylation using the specific EPHB4 kinase inhibitor NVP-BHG712 a decreased proliferation rate/vitality of synovial- and fibrosarcoma cells was observed. Silencing of EPHB4 significantly reduced the transmigration of synovial sarcoma cells towards fibroblasts and endothelial cells. In addition, we assessed the anti-metastatic effect of EPHB4 inhibition in vivo by intraperitoneal administration of the EPHB4 inhibitor in an appropriate sarcoma lung metastasis xenograft model. As result 43% of NVP-BHG712 treated mice (n = 3/7) developed pulmonary metastases whereas all control mice (n = 5) revealed lung metastases. The residual 57% of mice (n = 4/7) showed only small local tumor cell spots. Size measurements of the Vimentin positive area explained significant decrease in lung metastasis formation (p

We noted four cases of apparent in vivo emergence of teicoplanin resistance during failed therapy for initially teicoplanin-susceptible vanB vancomycin-resistant Enterococcus faecium (VREfm) infections in solid organ transplant recipients at our institution over a 12 month period. We investigated if in vivo emergence of resistance had occurred, if transplant-related vancomycin-resistant Enterococcus (VRE) infections had occurred and identified clinical predictors of resistance emergence.Whole genome sequencing was performed on nine VREfm isolates for phylogenetic analysis and to identify determinants of teicoplanin resistance. Clinical treatment details were compared with other patients who received teicoplanin for confirmed vanB VRE infections but did not develop resistance during the same year at our institution.A high-resolution, core genome phylogeny was inferred for nine VREfm isolates and confirmed in vivo development of resistance during failed therapy in four cases. Four different non-synonymous single nucleotide polymorphisms (SNPs) were observed in the vanRS genes of teicoplanin-resistant strains compared with the index teicoplanin-susceptible strains, and these SNPs were predicted to confer teicoplanin resistance. VREfm within a cluster of early transplant-related infections were phylogenetically identical at the core genome level, indicating a common source donor. Focus eradication and absence of prosthetic material were characteristics of those patients treated successfully.Clinicians should be cautious of resistance emerging during teicoplanin therapy for vanB VRE, particularly in immunosuppressed patients or where source control is difficult.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

To assess the cost-effectiveness of high-resolution anoscopy (HRA), anal cytology, and anal human papillomavirus (HPV) detection in screening for histologic high-grade anal intraepithelial neoplasia (AIN 2/3) in HIV-positive MSM.Participants were 401 HIV-positive MSM who were screened for anal cancer in a tertiary care HIV clinic.A decision analytical model was used to determine the cost-effectiveness of three anal cancer screening strategies: the direct use of HRA; HRA only if anal cytology was abnormal; and HRA only if oncogenic HPV was present. The model included the use of different thresholds for abnormal cytology and also combined cytology and HPV testing. The outcome was the number of AIN 2/3 cases detected. Costs were estimated from institutional data and sensitivity/specificity of cytology and HPV tests were obtained from the screening study.The costs ($ US) per procedure for HRA, cytology, and HPV testing were $193, $90, and $95, respectively. The direct use of HRA was the most cost-effective strategy. It detected 98 individuals with AIN 2/3 and had a cost-effectiveness of $809 per AIN 2/3 case detected. Using probabilistic sensitivity analysis, three other strategies had similar costs per case detected and might be as cost-effective as HRA.In HIV-infected MSM, the direct use of HRA is the most cost-effective strategy for detecting AIN 2/3. The higher cost per use for HRA was offset by the high sensitivity and low specificity of HPV and cytology testing.

We report radio-frequency (rf) electrodynamics in polycrystalline La0.67Ba0.23Ca0.1MnO3 as a function of temperature and magnetic field using a home-built LC resonant circuit powered by an integrated chip oscillator. The resonance frequency (fr) of the oscillator and the power (P) absorbed by the sample are measured simultaneously. The paramagnetic to ferromagnetic phase transition in the absence of an external magnetic field is accompanied by a rapid decrease in both P and fr around the Curie temperature TC=300 K. However, much below TC, the fr shows a step-like anomaly around 165 K (195 K) while cooling (warming), which we attribute to a structural phase transition from high temperature rhombohedral ( R 3 ¯ c ) to low temperature orthorhombic (Imma) phase. The step-like anomaly in fr versus T disappears in a field of 300 G. Fractional changes as large as 19% in Δfr/fr and 10% in ΔP/P are observed under H=1 kG around TC. Our study suggests that the rf resonance technique is a versatile tool to study the magnetization dynamics as well as to investigate the structural phase transition in manganites.

This study investigated the hypothesis that the main component character which contributes more to the whole-word meaning is used as an access code in reading. Grade 1, 3, and 5 readers were asked to read aloud two-character Chinese compound words (modifier, supplement, and coordinative words) with four patterns of component character frequency (high-high, high-low, low-high, and low-low). Results showed that the main component character effect was present for all three types of word structures, and the use of the main component character as a reading strategy was found in grade 3 and 5 children in their reading. It was proposed that representations of low-frequency Chinese compounds are stored in morphologically decomposed form and that word structure information has a role in the processing of compound words.