Core genome MLST results of <i>Paenibacillus larvae</i> isolates from the outbreak on Gotland in 2014.

<p>Two minimum spanning trees generated in SeqSphere+ based on two cgMLST-analysis with ERIC II-isolates (A) and ERIC I-isolates (B), respectively. Results in A are based on 3,309 target genes and B is based on 3,155 target genes. The numbers represent the number of allele differences between isolates. The branch lengths are not proportional to the number of differences. The colors indicate the beekeeper origin of the isolates.</p

Isolates included in the study, year of sampling, beekeeper, geographic and biological origin, ERIC-typing and 7-gene MSLT results.

<p>Isolates from Gotland are marked with the prefix “G” and isolates from Uppland with the prefix “U”.</p

A model for how heterogeneity is transmitted through the infectious dose.

<p>Colors represent different mutation profiles in a subclone (i.e., a subclonal mutation present in a fraction of the population). Heterogeneity in the preceding animal in the infectious chain may lead to different mutations being transmitted in different animals even though they were infected from the same carcass. The vertical black boxes represent the soil/sediment and can be seen as a randomizer mixing the spores which will infect next host. A clonal mutation (i.e., one present in the whole population) is valuable for tracing purposes but may be subclonal in the preceding case. Thus, it is evident that there is a need to analyze several isolates to determine the clonality of the observed mutations and to fully comprehend the transmission chain.</p

Microevolution during an Anthrax Outbreak Leading to Clonal Heterogeneity and Penicillin Resistance

<div><p>Anthrax is a bacterial disease primarily affecting grazing animals but it can also cause severe disease in humans. We have used genomic epidemiology to study microevolution of the bacterium in a confined outbreak in cattle which involved emergence of an antibiotic-resistant phenotype. At the time of death, the animals contained a heterogeneous population of Single Nucleotide Variants (SNVs), some being clonal but most being subclonal. We found that independent isolates from the same carcass had similar levels of SNV differences as isolates from different animals. Furthermore the relative levels of subclonal populations were different in different locations in the same carcass. The heterogeneity appeared to be derived in part from heterogeneity in the infectious dose. The resistance phenotype was linked to clonal mutations in an anti-sigma factor gene and in one case was preceded by an acquisition of a hypermutator phenotype. In another animal, small subclonal populations were observed with counteracting mutations that had turned off the resistance genes. In summary, this study shows the importance of accounting for both acquired and inherited heterogeneity when doing high-resolution infection tracing and when estimating the risks associated with penicillin treatment.</p></div

Signature analysis of Foot-and-Mouth Disease Virus (FMDV) serotypes.

<p>A fragmented alignment was performed with 50/25 settings using BLASTN (BLAST+). Target groups were formulated according to the serotype definitions. All other serotypes were used as background. The ‘maximum background/average target’ setting was used for biomarker score calculation. The annotations shown come from the type Asia 1 isolate IND 13–91(DQ989312). VP1–VP4 constitutes the capsid proteins that are exposed on the virus particle and are therefore important determinants for serotype classification.</p

Transcriptome analysis of genes involved in penicillin resistance.

<p>The three horizontal panels show reads for three samples mapped to the genome. The upper and middle panels show that the mutation in <i>rsiP</i> led to a profound up-regulation of five genes: A) <i>rsiP</i> anti-sigma factor (BAPAT_2394), <i>sigP</i> sigma factor (BAPAT_2393), beta-lactamase 1 (BAPAT_2397), penicillin-binding protein transpeptidase (BAPAT_2396) and shown in B) the beta-lactamase 2 (BAPAT_3350). The lower panel shows that the counteracting <i>sigP</i> mutation found in a subclonal population in one animal had quenched expression completely.</p

Overview of Gegenees.

<p>The Gegenees workspace contains one or several local databases. Genomes can be downloaded from the NCBI ftp site or from custom ftp sites through a built-in ftp client. This client compares the content of the local database with the remote one and highlights genomes already present locally. Unpublished genomes or genomes downloaded from other sources can be imported. The Gegenees workspace can also contain comparison projects. Genomes are added to the comparison from the local database. Genomes already in the active comparison are highlighted in the local database to facilitate the update process. Comparisons can also be downloaded from <a href="http://www.gegenees.org" target="_blank">www.gegenees.org</a> or shared between labs and imported into the workspace. One or several fragmented alignments can be made in the comparison with custom-specified resolution. Large alignments are associated with lengthy calculations and can therefore be paused and later resumed. Genomes can also later be added to a completed alignment that is then updated with the missing data points. When an alignment has been completed, the phylogenomic context can be analyzed in heat-plots. Nexus files can be exported for dendrogram construction and heat plots can be exported for high-resolution printouts. The alignment can then be analyzed in terms of Biomarker scores and uniqueness signatures. A target and a background group are defined on the basis of strain phenotypes and phylogenomic overview. The resulting conservation pattern signature can then be viewed and explored graphically or in tables. The signatures can also be exported to Artemis. Primers and/or probes can be designed from the signatures and candidate primers can be added back to Gegenees in form of a primer/probe alignment. Primer specificity can then be analyzed in terms of mismatches in the target and background groups.</p

Comparative analysis of the <i>Bacillus cereus</i> group.

<p>A heat-plot based on a 200/100 BLASTN fragmented alignment without threshold is shown. The figure is cropped to show only the <i>Bacillus cereus</i> group. Target groups used for PCR design are indicated (T1–T5). All remaining <i>Bacillus</i> genomes were used as a background group. This analysis was made without a threshold to filter non-conserved genetic material. Viewing the heat-plot without a threshold means that the values are based on both the core genome size and the core conservation. This often gives a better view during target group formulation because signatures are per definition outside the core when comparing a target genome with a background genome. Insert A shows the uniqueness signature for <i>B. anthracis</i> (T1). Signatures for all groups are present in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039107#pone.0039107.s005" target="_blank">Figure S3</a>. Insert B shows a dendrogram based on the heat plot. The dendrogram was produced in SplitsTree 4 (using neighbor joining method) made from a distance matrix Nexus file exported from Gegenees. <i>B. cytotoxicus</i> was set as outgroup.</p

Phylogenomic overview in Gegenees.

<p>Both heat-plots of the similarity matrices and trees created from the same data are shown. <b>A</b>. A Gegenees heat-plot over a set of <i>Bacillus</i> strains that had previously been analyzed by MLST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039107#pone.0039107-Kolsto1" target="_blank">[17]</a>. The heat-plot is based on a fragmented alignment using BLASTN made with settings 200/100. The cutoff threshold for non-conserved material was 30%. A dendrogram was produced in SplitsTree 4 (using neighbor joining method) made from a Nexus file exported from Gegenees. <i>B. cytotoxicus</i> was set as outgroup. The clustering is very similar to previously published trees. The scale bar represents a 1% difference in average BLASTN score similarity. <b>B</b>. A Gegenees heat-plot over a set of yeast genomes that has been analyzed before with different phylogenomic methods. These genomes are more distant from each other and a BLASTN comparison does not resolve them well (data not shown). A fragmented alignment in TBLASTX mode was performed with settings 200/200. The cutoff threshold for non-conserved material was 20%. A dendrogram was produced in SplitsTree 4 (using neighbor joining method) made from a distance matrix Nexus file exported from Gegenees. <i>Y. lipolytica</i> was set as outgroup. The clustering here is also very similar to the previously published trees <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039107#pone.0039107-Jeffroy1" target="_blank">[2]</a>. The scale bar represents a 10% difference in average TBLASTX score similarity.</p

The relationship between the mutational profiles of different isolates in this study.

<p>The dendrogram was made by identifying and concatenating all SNPs and indels in the chromosomes. The different colors represent different animals. For some animals, two independent isolates were recovered. The “Sediment” isolates were recovered from the presumed source of infection. Stars represent two different mutations in the gene <i>rsiP</i> that led to penicillin resistance. The Cow3Pc isolate showed 54 chromosomal mutations compared to Cow1 (indicated as a zigzag line). The black bar equals 1 bp difference.</p