Polymerase chain reaction for the in vitro detection of the pESI plasmid associated with the globally circulating Salmonella Infantis outbreak strain.

A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available.

Spontaneous Genomic Variation as a Survival Strategy of Nosocomial Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most important nosocomial human pathogens frequently isolated in bloodstream and medical device-related infections. However, its mechanisms of evolution and adaptation are still poorly explored. To characterize the strategies of genetic and phenotypic diversity in S. haemolyticus, we analyzed an invasive strain for genetic and phenotypic stability after serial passage in vitro in the absence and presence of beta-lactam antibiotics. We performed pulsed-field gel electrophoresis (PFGE) of the culture and analyzed five colonies at seven time points during stability assays for beta-lactam susceptibility, hemolysis, mannitol fermentation, and biofilm production. We compared their whole genomes and performed phylogenetic analysis based on core single-nucleotide polymorphisms (SNPs). We observed a high instability in the PFGE profiles at the different time points in the absence of antibiotic. Analysis of WGS data for individual colonies showed the occurrence of six large-scale genomic deletions within the oriC environ, smaller deletions in non-oriC environ regions, and nonsynonymous mutations in clinically relevant genes. The regions of deletion and point mutations included genes encoding amino acid and metal transporters, resistance to environmental stress and beta-lactams, virulence, mannitol fermentation, metabolic processes, and insertion sequence (IS) elements. Parallel variation was detected in clinically significant phenotypic traits such as mannitol fermentation, hemolysis, and biofilm formation. In the presence of oxacillin, PFGE profiles were overall stable over time and mainly corresponded to a single genomic variant. Our results suggest that S. haemolyticus populations are composed of subpopulations of genetic and phenotypic variants. The maintenance of subpopulations in different physiological states may be a strategy to adapt rapidly to stress situations imposed by the host, particularly in the hospital environment. IMPORTANCE The introduction of medical devices and antibiotics into clinical practice have substantially improved patient quality of life and contributed to extended life expectancy. One of its most cumbersome consequences was the emergence of medical device-associated infections caused by multidrug-resistant and opportunistic bacteria such as Staphylococcus haemolyticus. However, the reason for this bacterium's success is still elusive. We found that in the absence of environmental stresses, S. haemolyticus can spontaneously produce subpopulations of genomic and phenotypic variants with deletions/mutations in clinically relevant genes. However, when exposed to selective pressures, such as the presence of antibiotics, a single genomic variant will be recruited and become dominant. We suggest that the maintenance of these cell subpopulations in different physiological states is an extremely effective strategy to adapt to stresses imposed by the host or the infection environment and might contribute for S. haemolyticus survival and persistence in the hospital.

LMAS: evaluating metagenomic short de novo assembly methods through defined communities.

BACKGROUND: The de novo assembly of raw sequence data is key in metagenomic analysis. It allows recovering draft genomes from a pool of mixed raw reads, yielding longer sequences that offer contextual information and provide a more complete picture of the microbial community. FINDINGS: To better compare de novo assemblers for metagenomic analysis, LMAS (Last Metagenomic Assembler Standing) was developed as a flexible platform allowing users to evaluate assembler performance given known standard communities. Overall, in our test datasets, k-mer De Bruijn graph assemblers outperformed the alternative approaches but came with a greater computational cost. Furthermore, assemblers branded as metagenomic specific did not consistently outperform other genomic assemblers in metagenomic samples. Some assemblers still in use, such as ABySS, MetaHipmer2, minia, and VelvetOptimiser, perform relatively poorly and should be used with caution when assembling complex samples. Meaningful strain resolution at the single-nucleotide polymorphism level was not achieved, even by the best assemblers tested. CONCLUSIONS: The choice of a de novo assembler depends on the computational resources available, the replicon of interest, and the major goals of the analysis. No single assembler appeared an ideal choice for short-read metagenomic prokaryote replicon assembly, each showing specific strengths. The choice of metagenomic assembler should be guided by user requirements and characteristics of the sample of interest, and LMAS provides an interactive evaluation platform for this purpose. LMAS is open source, and the workflow and its documentation are available at https://github.com/B-UMMI/LMAS and https://lmas.readthedocs.io/, respectively.

Genome analysis of Legionella pneumophila ST23 from various countries reveals highly similar strains.

Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks.

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.

Chewie Nomenclature Server (chewie-NS): a deployable nomenclature server for easy sharing of core and whole genome MLST schemas.

Chewie Nomenclature Server (chewie-NS, https://chewbbaca.online/) allows users to share genome-based gene-by-gene typing schemas and to maintain a common nomenclature, simplifying the comparison of results. The combination between local analyses and a public repository of allelic data strikes a balance between potential confidentiality issues and the need to compare results. The possibility of deploying private instances of chewie-NS facilitates the creation of nomenclature servers with a restricted user base to allow compliance with the strictest data policies. Chewie-NS allows users to easily share their own schemas and to explore publicly available schemas, including informative statistics on schemas and loci presented in interactive charts and tables. Users can retrieve all the information necessary to run a schema locally or all the alleles identified at a particular locus. The integration with the chewBBACA suite enables users to directly upload new schemas to chewie-NS, download existing schemas and synchronize local and remote schemas from chewBBACA command line version, allowing an easier integration into high-throughput analysis pipelines. The same REST API linking chewie-NS and the chewBBACA suite supports the interaction of other interfaces or pipelines with the databases available at chewie-NS, facilitating the reusability of the stored data.

Distance-based phylogenetic inference from typing data: a unifying view.

Typing methods are widely used in the surveillance of infectious diseases, outbreaks investigation and studies of the natural history of an infection. Moreover, their use is becoming standard, in particular with the introduction of high-throughput sequencing. On the other hand, the data being generated are massive and many algorithms have been proposed for a phylogenetic analysis of typing data, addressing both correctness and scalability issues. Most of the distance-based algorithms for inferring phylogenetic trees follow the closest pair joining scheme. This is one of the approaches used in hierarchical clustering. Moreover, although phylogenetic inference algorithms may seem rather different, the main difference among them resides on how one defines cluster proximity and on which optimization criterion is used. Both cluster proximity and optimization criteria rely often on a model of evolution. In this work, we review, and we provide a unified view of these algorithms. This is an important step not only to better understand such algorithms but also to identify possible computational bottlenecks and improvements, important to deal with large data sets.

Two multi-fragment recombination events resulted in the ß-lactam-resistant serotype 11A-ST6521 related to Spain9V-ST156 pneumococcal clone spreading in south-western Europe, 2008 to 2016.

BackgroundThe successful pneumococcal clone Spain9V-ST156 (PMEN3) is usually associated with vaccine serotypes 9V and 14.AimOur objective was to analyse the increase of a serotype 11A variant of PMEN3 as cause of invasive pneumococcal disease (IPD) in Spain and its spread in south-western Europe.MethodsWe conducted a prospective multicentre study of adult IPD in Spain (2008-16). Furthermore, a subset of 61 penicillin-resistant serotype 11A isolates from France, Italy, Portugal and Spain were subjected to whole genome sequencing (WGS) and compared with 238 genomes from the European Nucleotide Archive (ENA).ResultsAlthough the incidence of serotype 11A in IPD was stable, a clonal shift was detected from CC62 (penicillin-susceptible) to CC156 (penicillin-resistant). By WGS, three major 11A-CC156 lineages were identified, linked to ST156 (n = 5 isolates; France, Italy and Portugal), ST166 (n = 4 isolates; France and Portugal) and ST838/6521 (n = 52 isolates; France, Portugal and Spain). Acquisition of the 11A capsule allowed to escape vaccine effect. AP200 (11A-ST62) was the donor for ST156 and ST838/6521 but not for ST166. In-depth analysis of ST838/6521 lineage showed two multi-fragment recombination events including four and seven fragments from an 11A-ST62 and an NT-ST344 representative, respectively.ConclusionThe increase in penicillin-resistant serotype 11A IPD in Spain was linked to the spread of a vaccine escape PMEN3 recombinant clone. Several recombination events were observed in PMEN3 acquiring an 11A capsule. The most successful 11A-PMEN3 lineage spreading in south-western Europe appeared after two multi-fragment recombination events with representatives of two major pneumococcal clones (11A-ST62 and NT-ST344).

DEN-IM: dengue virus genotyping from amplicon and shotgun metagenomic sequencing.

Dengue virus (DENV) represents a public health threat and economic burden in affected countries. The availability of genomic data is key to understanding viral evolution and dynamics, supporting improved control strategies. Currently, the use of high-throughput sequencing (HTS) technologies, which can be applied both directly to patient samples (shotgun metagenomics) and to PCR-amplified viral sequences (amplicon sequencing), is potentially the most informative approach to monitor viral dissemination and genetic diversity by providing, in a single methodological step, identification and characterization of the whole viral genome at the nucleotide level. Despite many advantages, these technologies require bioinformatics expertise and appropriate infrastructure for the analysis and interpretation of the resulting data. In addition, the many software solutions available can hamper the reproducibility and comparison of results. Here we present DEN-IM, a one-stop, user-friendly, containerized and reproducible workflow for the analysis of DENV short-read sequencing data from both amplicon and shotgun metagenomics approaches. It is able to infer the DENV coding sequence (CDS), identify the serotype and genotype, and generate a phylogenetic tree. It can easily be run on any UNIX-like system, from local machines to high-performance computing clusters, performing a comprehensive analysis without the requirement for extensive bioinformatics expertise. Using DEN-IM, we successfully analysed two types of DENV datasets. The first comprised 25 shotgun metagenomic sequencing samples from patients with variable serotypes and genotypes, including an in vitro spiked sample containing the four known serotypes. The second consisted of 106 paired-end and 76 single-end amplicon sequences of DENV 3 genotype III and DENV 1 genotype I, respectively, where DEN-IM allowed detection of the intra-genotype diversity. The DEN-IM workflow, parameters and execution configuration files, and documentation are freely available at https://github.com/B-UMMI/DEN-IM).

Lactobacillus mulieris sp. nov., a new species of Lactobacillus delbrueckii group.

One Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161MT, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161MT belonged to the genus Lactobacillus. Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161MT and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii, respectively. The major fatty acids of strain c10Ua161MT were C18 : 1ω9c (65.0%), C16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C18 : 1ω7c, and/or C18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161MT represents a novel species of the genus Lactobacillus, for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161MT (=CECT 9755T=DSM 108704T).

BacPipe: A Rapid, User-Friendly Whole-Genome Sequencing Pipeline for Clinical Diagnostic Bacteriology.

Despite rapid advances in whole genome sequencing (WGS) technologies, their integration into routine microbiological diagnostics has been hampered by the lack of standardized downstream bioinformatics analysis. We developed a comprehensive and computationally low-resource bioinformatics pipeline (BacPipe) enabling direct analyses of bacterial whole-genome sequences (raw reads or contigs) obtained from second- or third-generation sequencing technologies. A graphical user interface was developed to visualize real-time progression of the analysis. The scalability and speed of BacPipe in handling large datasets was demonstrated using 4,139 Illumina paired-end sequence files of publicly available bacterial genomes (2.9-5.4 Mb) from the European Nucleotide Archive. BacPipe is integrated in EBI-SELECTA, a project-specific portal (H2020-COMPARE), and is available as an independent docker image that can be used across Windows- and Unix-based systems. BacPipe offers a fully automated "one-stop" bacterial WGS analysis pipeline to overcome the major hurdle of WGS data analysis in hospitals and public-health and for infection control monitoring.

Heterogeneity of penicillin-non-susceptible group B streptococci isolated from a single patient in Germany.

OBJECTIVES: Streptococcus agalactiae [group B streptococci (GBS)] have been considered uniformly susceptible to penicillin. However, increasing reports from Asia and North America are documenting penicillin-non-susceptible GBS (PRGBS) with mutations in pbp genes. Here we report, to the best of our knowledge, the first two PRGBS isolates recovered in Europe (AC-13238-1 and AC-13238-2), isolated from the same patient. METHODS: Two different colony morphologies of GBS were noted from a surgical abscess drainage sample. Both were serotyped and antimicrobial susceptibility testing was performed by different methodologies. High-throughput sequencing was done to compare the isolates at the genomic level, to identify their capsular type and ST, to evaluate mutations in the pbp genes and to compare the isolates with the genomes of other PRGBS isolates sharing the same serotype and ST. RESULTS: Isolates AC-13238-1 and AC-13238-2 presented MICs above the EUCAST and CLSI breakpoints for penicillin susceptibility. Both shared the capsular type Ia operon and ST23. Genomic analysis uncovered differences between the two isolates in seven genes, including altered pbp genes. Deduced amino acid sequences revealed critical substitutions in PBP2X in both isolates. Comparison with serotype Ia clonal complex 23 PRGBS from the USA reinforced the similarity between AC-13238-1 and AC-13238-2, and their divergence from the US strains. CONCLUSIONS: Our results support the in-host evolution of ß-lactam-resistant GBS, with two PRGBS variants being isolated from one patient.

Natural Occurrence of Escherichia coli-Infecting Bacteriophages in Clinical Samples.

The interaction between bacteriophages, bacteria and the human host as a tripartite system has recently captured attention. The taxonomic diversity of bacteriophages, as a natural parasite of bacteria, still remains obscure in human body biomes, representing a so-called "viral dark matter." Here, we isolated and characterized coliphages from blood, urine and tracheal aspirates samples collected at a tertiary care hospital in Austria. Phages were more often isolated from blood, followed by urine and tracheal aspirates. Phylogenetic analysis and genome comparisons allowed the identification of phages belonging to the Tunavirinae subfamily, and to the Peduovirus and Tequintavirus genera. Tunavirinae phages cluster together and are found in samples from 14 patients, suggesting their prevalence across a variety of human samples. When compared with other phage genomes, the highest similarity level was at 87.69% average nucleotide identity (ANI), which suggests that these are in fact a newly isolated phage species. Tequintavirus phages share a 95.90% with phage 3_29, challenging the ANI threshold currently accepted to differentiate phage species. The isolated phages appear to be virulent, with the exception of the Peduovirus members, which are integrative and seem to reside as prophages in bacterial genomes.

Draft Genome Sequence of Escherichia coli DSM 12242, a Highly Efficient Host Strain for the Isolation of Somatic Coliphages.

Here, we report the draft genome sequence and characterization of the commercial strain Escherichia coli DSM 12242 (=ATCC 13706/60 =NZRM 3262) derived from strain C (ATCC 13706), which is suitable for the isolation of coliphages from environmental and clinical samples.

Distinct Phenotypic and Genomic Signatures Underlie Contrasting Pathogenic Potential of Staphylococcus epidermidis Clonal Lineages.

Background: Staphylococcus epidermidis is a common skin commensal that has emerged as a pathogen in hospitals, mainly related to medical devices-associated infections. Noteworthy, infection rates by S. epidermidis have the tendency to rise steeply in next decades together with medical devices use and immunocompromized population growth. Staphylococcus epidermidis population structure includes two major clonal lineages (A/C and B) that present contrasting pathogenic potentials. To address this distinction and explore the basis of increased pathogenicity of A/C lineage, we performed a detailed comparative analysis using phylogenetic and integrated pangenome-wide-association study (panGWAS) approaches and compared the lineages's phenotypes in in vitro conditions mimicking carriage and infection. Results: Each S. epidermidis lineage had distinct phenotypic signatures in skin and infection conditions and differed in genomic content. Combination of phenotypic and genotypic data revealed that both lineages were well adapted to skin environmental cues. However, they appear to occupy different skin niches, perform distinct biological functions in the skin and use different mechanisms to complete the same function: lineage B strains showed evidence of specialization to survival in microaerobic and lipid rich environment, characteristic of hair follicle and sebaceous glands; lineage A/C strains showed evidence for adaption to diverse osmotic and pH conditions, potentially allowing them to occupy a broader and more superficial skin niche. In infection conditions, A/C strains had an advantage, having the potential to bind blood-associated host matrix proteins, form biofilms at blood pH, resist antibiotics and macrophage acidity and to produce proteases. These features were observed to be rare in the lineage B strains. PanGWAS analysis produced a catalog of putative S. epidermidis virulence factors and identified an epidemiological molecular marker for the more pathogenic lineage. Conclusion: The prevalence of A/C lineage in infection is probably related to a higher metabolic and genomic versatility that allows rapid adaptation during transition from a commensal to a pathogenic lifestyle. The putative virulence and phenotypic factors associated to A/C lineage constitute a reliable framework for future studies on S. epidermidis pathogenesis and the finding of an epidemiological marker for the more pathogenic lineage is an asset for the management of S. epidermidis infections.

Author Correction: Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Streptococcus pneumoniae Serotype 3 in Mexico (1994 to 2017): Decrease of the Unusual Clonal Complex 4909 Lineage following PCV13 Introduction.

Streptococcus pneumoniae expressing serotype 3 has a high virulence and a high case fatality ratio. Most studies of serotype 3 pneumococci have focused on a single lineage, the widespread sequence type 180 (ST180). To evaluate the serotype 3 lineages causing infections in Mexico, we characterized 196 isolates recovered from 1994 to 2017. The isolates were mostly susceptible to all antimicrobials tested. A single meningitis isolate was resistant to penicillin, and the resistance to erythromycin was 5.2%. The isolates represented the widely disseminated clonal complex 180 (CC180; n = 140), the unusual CC4909 (n = 42), CC260 (n = 11), and a few singletons (n = 3). CC260 was less frequent among pneumococcal invasive disease isolates than CC180 and CC4909 (P = 0.015). There was a decrease of CC4909 (P < 0.001) following PCV13 introduction (2012 to 2017). The CC4909 isolates were represented mostly by ST1119 (n = 40), seemingly having a restricted geographic origin, with isolates in the PubMLST database having been recovered only in Mexico, the United States, and Germany. A genomic analysis of publicly available genomes showed that ST1119 isolates have less than 32% similarity with ST180 isolates, indicating that these lineages are more separated than revealed by traditional multilocus sequence typing. Considering the suggestions of a lower efficacy of the 13-valent pneumococcal conjugate vaccine against serotype 3, the different dynamics of the two major serotype 3 lineages in Mexico following the introduction of PCV13 should be closely monitored.

Plasmid ATLAS: plasmid visual analytics and identification in high-throughput sequencing data.

Plasmid ATLAS (pATLAS, http://www.patlas.site) provides an easy-to-use web accessible database with visual analytics tools to explore the relationships of plasmids available in NCBI's RefSeq database. pATLAS has two main goals: (i) to provide an easy way to search for plasmids deposited in NCBI RefSeq and their associated metadata; (ii) to visualize the relationships of plasmids in a graph, allowing the exploration of plasmid evolution. pATLAS allows searching by plasmid name, bacterial host taxa, antibiotic resistance and virulence genes, plasmid families, and by sequence length and similarity. pATLAS is also able to represent in the plasmid network, plasmid sets identified by external pipelines using mapping, mash screen or assembly from high-throughput sequencing data. By representing the identified hits within the network of relationships between plasmids, allowing the possibility of removing redundant results, and by taking advantage of the browsing capabilities of pATLAS, users can more easily interpret the pipelines' results. All these analyses can be saved to a JSON file for sharing and future re-evaluation. Furthermore, by offering a REST-API, the pATLAS database and network display are easily accessible by other interfaces or pipelines.

Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.

GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens.

Current methods struggle to reconstruct and visualize the genomic relationships of large numbers of bacterial genomes. GrapeTree facilitates the analyses of large numbers of allelic profiles by a static "GrapeTree Layout" algorithm that supports interactive visualizations of large trees within a web browser window. GrapeTree also implements a novel minimum spanning tree algorithm (MSTree V2) to reconstruct genetic relationships despite high levels of missing data. GrapeTree is a stand-alone package for investigating phylogenetic trees plus associated metadata and is also integrated into EnteroBase to facilitate cutting edge navigation of genomic relationships among bacterial pathogens.