Species identification and genotyping of Citrobacter spp. using genes with high nucleotide diversity.

Citrobacter spp. are facultative anaerobic Gram-negative bacillus found in a wide range of habitats. Recently, there have been reports of an increasing number of cases of Citrobacter spp. being the cause of nosocomial infections, and of increasing highly antimicrobial-resistant (AMR) Citrobacter spp. In clinical laboratory testing, Citrobacter spp., such as C. freundii and C. braakii, are handled collectively as the C. freundii complex. This can be obstacle to collect information regarding species identification and genetic lineage which are necessary for estimating distribution mechanism and evaluating AMR degree for each Citrobacter species. This study investigated gene combinations that allow for Citrobacter spp. identification and genetic lineage estimation with as few genes as possible. Citrobacter genomes of 18 species were collected as many as possible from GenBank. After the gene annotation, nucleotide diversity of 1,113 genes contained in all 453 genomes was calculated. Genotypes of top seven highest nucleotide diversity (HND) genes were confirmed and investigated their distributions among Citrobacter genomes. Except for C. werkmanii and C. cronae, it was suggested that the genotype of the top HND gene, groups_3152, and combinations of the genotypes of the top two HND genes were a species- and genetic lineage-specific distribution, respectively. Considering that the genomes of C. werkmanii and C. cronae were highly similar and indistinguishable each other by conventional genotyping methods, it was suggested that the combination of the top two HND genes could be used to Citrobacter spp. identification and genetic lineage classification. Importance StatementMolecular epidemiology of individual Citrobacter species has not been widely performed, partly because Citrobacter spp. are handled as the C. freundii complex in the clinical testing and the multi-locus sequencing typing (MLST) has been performed targeting the entire genus Citrobacter. This study proposed a combination of two genes for identification of Citrobacter spp. and classify their genetic lineages. Currently, phylogenetic tree analysis has become possible using core-genome MLST and core-genome single nucleotide polymorphisms (SNPs) after whole genome sequencing (WGS) of bacterial isolates. But these phylogenetic analyses require a considerable amount of cost. In this regard, using two genes for Citrobacter species identification and genetic lineage classification reduces the required cost compared to WGS, encouraging its introduction into clinical testing and further performing molecular epidemiology of individual Citrobacter species.