| Summary: | The genetic heterogeneity of <i>Heyndrickxia sporothermodurans</i> (formerly <i>Bacillus</i><i>sporothermodurans)</i> was evaluated using whole genome sequencing. The genomes of 29 previously identified <i>Heyndrickxia</i><i>sporothermodurans</i> and two <i>Heyndrickxia vini</i> strains isolated from ultra-high-temperature (UHT)-treated milk were sequenced by short-read (Illumina) sequencing. After sequence analysis, the two <i>H. vini</i> strains could be reclassified as <i>H. sporothermodurans</i>. In addition, the genomes of the <i>H.</i><i>sporothermodurans</i> type strain (DSM 10599<sup>T</sup>) and the closest phylogenetic neighbors <i>Heyndrickxia</i><i>oleronia</i> (DSM 9356<sup>T</sup>) and <i>Heyndrickxia vini</i> (JCM 19841<sup>T</sup>) were also sequenced using both long (MinION) and short-read (Illumina) sequencing. By hybrid sequence assembly, the genome of the <i>H. sporothermodurans</i> type strain was enlarged by 15% relative to the short-read assembly. This noticeable increase was probably due to numerous mobile elements in the genome that are presumptively related to spore heat tolerance. Phylogenetic studies based on 16S rDNA gene sequence, core genome, single-nucleotide polymorphisms and ANI/dDDH, showed that <i>H. vini</i> is highly related to <i>H. sporothermodurans</i>. When examining the genome sequences of all <i>H.</i><i>sporothermodurans</i> strains from this study, together with 4 <i>H. sporothermodurans</i> genomes available in the GenBank database, the majority of the 36 strains examined occurred in a clonal lineage with less than 100 SNPs. These data substantiate previous reports on the existence and spread of a genetically highly homogenous and heat resistant spore clone, i.e., the HRS-clone.
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