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The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http:// www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) communityoriented database infrastructure and analysis tools. IMPORTANCE The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine wholegenome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets.

Staphylococcus aureus ist ein pathogenes Bakterium, das eine Vielzahl verschiedener Krankheiten auslösen kann. Eine Bedrohung geht von Methicillin-resistenten S. aureus aus, die Resistenzen gegen Antibiotika entwickelt haben. In dieser Arbeit wurden neue Sequenziertechnologien verwendet, um durch die Sequenzierung ausgewählter MRSA-Genome Einblicke in die Evolution und die räumliche Ausbreitung dieses Keims zu gewinnen. Der Vergleich von 24 MRSA-Genomen des klonalen Komplexes CC5 zeigt wenig Variabilität im Kerngenom, aber eine hohe Diversität in Prophagen. Auf der Basis Genom-weiter SNPs wurde die Phylogenie von CC5 mit der zurzeit höchsten Auflösung rekonstruiert. Durch die Abbildung der Prophagen auf die Phylogenie der Wirts-Staphylokokken konnte gezeigt werden, dass der Austausch von Phagensequenzen eine wichtige Rolle für die Evolution von MRSA spielt. Durch Rekombination akkumulieren die Prophagen offenbar kontinuierlich Sequenzunterschiede, so dass ihre Diversität mit der phylogenetischen Distanz zwischen den Wirtsbakterien zunimmt. In dieser Arbeit konnte erstmals gezeigt werden, dass MRSA-Genome genügend Informationen enthalten, um auch für Epidemiestämme die Übertragung des Erregers zwischen einzelnen Patienten während eines Krankenhaus-Aufenthalts nachzuweisen. Darüber hinaus konnte durch eine phylogenetische Analyse sogar die Richtung der Transmission von einem Patienten auf einen anderen bestimmt werden. Weiterhin konnte die Evolution von MRSA innerhalb einzelner Patienten beobachtet werden. Die Isolate zeigten zwar eine über die Zeit ansteigende, genetische Distanz zu dem jeweils infizierenden Stamm, ihre Diversität nahm jedoch nicht zu. Vielmehr war jeder Patient zu jedem Zeitpunkt mit einem einzelnen, genetisch homogenen Klon besiedelt. Sequenzen ganzer Genome ermöglichen es, die Evolution von Pathogenen mit hoher Auflösung zu untersuchen. In naher Zukunft wird die Sequenzierung ganzer Genome eine wichtige Rolle in der mikrobiellen Diagnostik spielen., Staphylococcus aureus is a major pathogen that causes a variety of infections. A particular danger originates from methicillin-resistant S. aureus (MRSA) which have become resistant to a wide spectrum of antibiotics. For the present work novel sequencing technologies were applied to sequence selected MRSA genomes to get deeper insights into the evolution and the spatial spread of this particular pathogen. The comparison of 24 MRSA genomes of the clonal complex CC5 revealed little variability within the core genome itself but a high level of diversity within mobile genetic elements, especially prophages. On the basis of genome wide SNPs the phylogeny of the clonal complex CC5 was reconstructed to the highest possible degree. Through the mapping of the prophages onto the phylogenetic tree of the host Staphylococci it could be shown that the exchange of phage sequences plays an important role in the short-term evolution of MRSA. Prophages accumulate sequence differences continuously through homologous recombination, causing an increase in their diversity with the phylogenetic distance between the host bacteria. This work was able to show for the first time that genome sequences from highly prevalent, epidemic MRSA contain enough information to verify the transmission of the pathogen between patients during hospitalisation. The use of phylogentic methods was successful in determining the direction of transmission from one patient to another. Furthermore, the evolution of MRSA within unique patients was investigated. The isolates showed an increasing genetic distance to the infecting strain but at the time their diversity did not rise. In fact each patient was colonised with a unique genetically homogeneous clone. Whole genome sequencing provides a detailed insight into the evolution of a pathogen at a high resolution. In the near future the sequencing of whole genomes will play a major role in microbial diagnostic.

Supplementary materials. Description: PDF file containing Figures S1-S10, and Tables S1 and Table S3. (PDF 14325 kb)

Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae. Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of colR in K. pneumoniae are largely due to chromosomal mutations in two-component regulators, although a plasmid-mediated colR mechanism has recently been uncovered. However, the effects of intrinsic colistin resistance are yet to be characterized on a whole-genome level. Here, we used a genomics-based approach to understand the mechanisms of adaptive colR acquisition in K. pneumoniae. In controlled directed-evolution experiments we observed two distinct paths to colistin resistance acquisition. Whole genome sequencing identified mutations in two colistin resistance genes: in the known colR regulator phoQ which became fixed in the population and resulted in a single amino acid change, and unstable minority variants in the recently described two-component sensor crrB. Through RNAseq and microscopy, we reveal the broad range of effects that colistin exposure has on the cell. This study is the first to use genomics to identify a population of minority variants with mutations in a colR gene in K. pneumoniae., This work was supported by the Wellcome Trust grant number WT098051. The salaries of AKC and CJB were supported by the Medical Research Council [grant number G1100100/1] and MJE is supported by Public Health England. LB is supported by a research fellowship from the Alexander von Humboldt Stiftung/Foundation. KEH is supported by the NHMRC of Australia (Fellowship #1061409).

The increasing incidence and emergence of multi-drug resistant (MDR) Acinetobacter baumannii has become a major global health concern. Colistin is a historic antimicrobial that has become commonly used as a treatment for MDR A. baumannii infections. The increase in colistin usage has been mirrored by an increase in colistin resistance. We aimed to identify the mechanisms associated with colistin resistance in A. baumannii using multiple high-throughput-sequencing technologies, including transposon-directed insertion site sequencing (TraDIS), RNA sequencing (RNAseq) and whole-genome sequencing (WGS) to investigate the genotypic changes of colistin resistance in A. baumannii. Using TraDIS, we found that genes involved in drug efflux (adeIJK), and phospholipid (mlaC, mlaF and mlaD) and lipooligosaccharide synthesis (lpxC and lpsO) were required for survival in sub-inhibitory concentrations of colistin. Transcriptomic (RNAseq) analysis revealed that expression of genes encoding efflux proteins (adeI, adeC, emrB, mexB and macAB) was enhanced in in vitro generated colistin-resistant strains. WGS of these organisms identified disruptions in genes involved in lipid A (lpxC) and phospholipid synthesis (mlaA), and in the baeS/R two-component system (TCS). We additionally found that mutations in the pmrB TCS genes were the primary colistin-resistance-associated mechanisms in three Vietnamese clinical colistin-resistant A. baumannii strains. Our results outline the entire range of mechanisms employed in A. baumannii for resistance against colistin, including drug extrusion and the loss of lipid A moieties by gene disruption or modification.

Supplementary materials. Description: PDF file containing Figures S1-S10, and Tables S1 and Table S3. (PDF 14325 kb)