Ribosomal MLST nucleotide identity (rMLST-NI), a rapid bacterial species identification method: application to Klebsiella and Raoultella genomic species validation

Abstract

Bacterial genomics is making an increasing contribution to the fields of medicine and public health microbiology. Consequently, accurate species identification of bacterial genomes is an important task, particularly as the number of genomes stored in online databases increases rapidly and new species are frequently discovered. Existing database entries require regular re-evaluation to ensure that species annotations are consistent with the latest species definitions. We have developed an automated method for bacterial species identification that is an extension of ribosomal multilocus sequence typing (rMLST). The method calculates an ‘rMLST nucleotide identity’ (rMLST-NI) based on the nucleotides present in the protein-encoding ribosomal genes derived from bacterial genomes. rMLST-NI was used to validate the species annotations of 11839 publicly available Klebsiella and Raoultella genomes based on a comparison with a library of type strain genomes. rMLST-NI was compared with two whole-genome average nucleotide identity methods (OrthoANIu and FastANI) and the k-mer based Kleborate software. The results of the four methods agreed across a dataset of 11839 bacterial genomes and identified a small number of entries (n=89) with species annotations that required updating. The rMLST-NI method was 3.5 times faster than Kleborate, 4.5 times faster than FastANI and 1600 times faster than OrthoANIu. rMLST-NI represents a fast and generic method for species identification using type strains as a reference.

Two novel Raoultella species associated with bleeding cankers of broadleaf hosts, Raoultella scottia sp. nov. and Raoultella lignicola sp. nov.

“…Results from the phylogenomic analyses agree with those of Liu et al (2020) , with species of the genus Klebsiella divided into two clades and Raoultella species constituting their own discrete clade. Furthermore, the topology of the phylogenomic tree is congruent with a previous phylogeny based on non-paralogous ribosomal MLST loci ( Bray et al, 2022 ) where the genus Raoultella is shown to separate Klebsiella into two clades, the “ pneumoniae-africana-variicola ” clade and the “ oxytoca-indica-grimontii-michiganensis ” clade.…”

“…The future of the genus Raoultella is currently unclear as taxonomists fail to agree on whether the genus Klebsiella is polyphyletic, or monophyletic with the inclusion of Raoultella in the Klebsiella genus clade. There is little to differentiate these two genera based on phenotype or ecology, and their phylogeny varies depending on whether it is based on core genome sequences, core protein sequences or rMLST loci ( Liu et al, 2020 ; Ma et al, 2021 ; Bray et al, 2022 ). As additional novel species are classified as belonging to either Raoultella or Klebsiella , the true taxonomy of these genera may become more apparent.…”

Rapid identification of enteric bacteria from whole genome sequences using average nucleotide identity metrics

“…Ribosomal MLST was described by Jolley et al (2012) , but this method requires gene prediction, unlike ANIm and FastANI ( Jolley et al, 2012 ). More recently, a new method for ribosomal MLST nucleotide identity (r-MLST-NI) has been developed for classifying Klebsiella and Raoultella species and may be useful for identifying other bacterial species ( Bray et al, 2022 ). Public health laboratories in the United States, including our laboratory, have transitioned to WGS analysis from conventional methods for identification and surveillance of enteric pathogens.…”