Short-Read Genome Sequencing, Analysis, and Precise Species Identification

All strains underwent whole-genome sequencing by using the HiSeq X10 platform (Illumina, https://www.illumina.com). Genomic DNA was prepared by using the QIAamp DNA mini kit (QIAGEN, https://www.qiagen.com). We used Unicycler version 0.4.3 (15), in the conservative mode for increased accuracy, to perform a de novo hybrid assembly. Precise species identification was established by determining the pairwise isDDH between the genome sequence of the query strain and those of type strains of Enterobacter spp., including the validly published species and the species awaiting validation (Appendix 1 Table 1). This process was performed by using the Genome-to-Genome Distance Calculator, formula 2 (16). A >70% cutoff was applied to define a species. In addition, we determined the pairwise ANI of the genome sequence of the query strain and those of type strains of Enterobacter spp. (Appendix 1 Table 1) by using JSpecies software (https://imedea.uib-csic.es/jspecies) with a >96% ANI cutoff to define a species (10). Sequence types (STs) were determined by using the genomic sequences to query the multilocus sequence typing database of E. cloacae (https://pubmlst.org/ecloacae). Antimicrobial resistance genes were identified from genome sequences by using the ABRicate program (https://github.com/tseemann/abricate) to query the ResFinder database (https://genomicepidemiology.org).

The genome sequences of all Enterobacter strains recovered from human blood (n = 349, Appendix 2 Table 2) were retrieved from GenBank (accessed 2018 Nov 1). These Enterobacter genomes were subjected to precise species identification by using the Genome-to-Genome Distance Calculator as described.

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