Revisiting Francisella tularensis subsp. holarctica, Causative Agent of Tularemia in Germany With Bioinformatics: New Insights in Genome Structure, DNA Methylation and Comparative Phylogenetic Analysis

Abstract

Francisella (F.) tularensis is a highly virulent, Gram-negative bacterial pathogen and the causative agent of the zoonotic disease tularemia. Here, we generated, analyzed and characterized a high quality circular genome sequence of the F. tularensis subsp. holarctica strain 12T0050 that caused fatal tularemia in a hare. Besides the genomic structure, we focused on the analysis of oriC, unique to the Francisella genus and regulating replication in and outside hosts and the first report on genomic DNA methylation of a Francisella strain. The high quality genome was used to establish and evaluate a diagnostic whole genome sequencing pipeline. A genotyping strategy for F. tularensis was developed using various bioinformatics tools for genotyping. Additionally, whole genome sequences of F. tularensis subsp. holarctica isolates isolated in the years 2008–2015 in Germany were generated. A phylogenetic analysis allowed to determine the genetic relatedness of these isolates and confirmed the highly conserved nature of F. tularensis subsp. holarctica.

Complete genome and plasmid sequences of a multidrug-resistant Salmonella enterica subsp. enterica serovar Panama isolate from a German cattle farm

“…Reads were de novo assembled using SPAdes 3.12.0 in Bayes Hammer mode (--careful) 13 and evaluated with QUAST v4.3 14 in standard setting. The filtering of the sample was performed by removing contigs with coverage less than 5× and size below 500 bases as described before 15 . ResFinder 16 , PlasmidFinder 17 , SeqSero 18 , SPIFinder 19 and its databases were accessed through the Center of Genomic Epidemiology ( http://www.genomicepidemiology.org/ , accessed January 2020).…”

Using affinity propagation clustering for identifying bacterial clades and subclades with whole-genome sequences of Francisella tularensis

“…For this study, F. tularensis subsp. holarctica isolates collected in the years 2006-2018 were used and are in part publically available [1,9,11,[30][31][32][33][34]. All isolates were manually checked for completeness of metadata, e.g.…”

“…Only assemblies without possible contamination or incomplete sequencing were allowed by excluding assemblies >4 Mb and <1 Mb of predicted total length. TempEst was used for the visualization and analysis of temporally sampled sequence data [41] as specified before [11].…”

“…An analysis with BEAST, as done before with only 67 samples [2] is not recommended, because the molecular clock models employed are statistically conditioned on having an evolutionary rate greater than zero [41]. That might be due to fewer samples or to a different assembler strategy, that had been shown to an over-and underestimated gene content and genome sizes up to 36% [11].…”

“…holarctica is endemic in Europe [ 2 , 8 ]. In Germany and France, most human infections are caused by contact with infected European brown hares ( Lepus europaeus ) [ 1 , 9 – 11 ].…”