Reclassification of Catabacter hongkongensis as Christensenella hongkongensis comb. nov. based on whole genome analysis

The genera Catabacter (family ‘Catabacteraceae’) and Christensenella (family Christensenellaceae ) are close relatives within the phylum Firmicutes . Members of these genera are strictly anaerobic, non-spore-forming and short straight rods with diverse phenotypes. Phylogenetic analysis of 16S rRNA genes suggest that Catabacter splits Christensenella into a polyphyletic clade. In an effort to ensure that family/genus names represent monophyletic clades, we performed a whole-genome based analysis of the genomes available for the cultured representatives of these genera: four species of Christensenella and two strains of Catabacter hongkongensis . A concatenated alignment of 135 shared protein sequences of single-copy core genes present in the included strains indicates that C. hongkongensis is indeed nested within the Christensenella clade. Based on their evolutionary relationship, we propose the transfer of Catabacter hongkongensis to the genus Christensenella as Christensenella hongkongensis comb. nov.


INTRODUCTION
Catabacter hongkongensis was first isolated in 2007 from the blood cultures of four patients in Hong Kong and Canada. Based on the phylogenetic positioning of 16S rRNA gene sequences and phenotypic characteristics, it was proposed as a new genus and new family, 'Catabacteraceae' [1]. The genus Catabacter comprises just one species, with the type strain Catabacter hongkongensis HKU16 T . Based on 16S rRNA gene sequencing surveys, C. hongkongensis has been detected in the blood of patients with diseases such as intestinal obstruction, gastrointestinal malignancy, acute cholecystitis and hypertension in Europe, North America and Asia [1][2][3][4][5]. Although Catabacter hongkongensis was first identified in 2007, the name Catabacter hongkongensis was validly published in 2014 [6].
16S rRNA gene sequence identity (%ID) has been used to delineate genus (95 %ID) and species (98.7 %ID) cutoffs [11,12]. The 16S rRNA gene sequence of C. hongkongensis HKU16 T has 96-97 %ID with the 16S rRNA genes of the four species of Christensenella, which places them in the range of sharing a genus using that criterion. In addition to sequence similarity, the 16S rRNA gene-based phylogenetic relationships of these taxa indicate they form a monophyletic clade [13].
Whole genome-based analysis with concatenated protein sequences has recently been proposed as a basis for determining the phylogenetic relationships of members of the Bacteria and Archaea [14]. Based on whole genome comparisons, Catabacter and Christensenella were annotated as belonging to the family Christensenellaceae in the order Christensenellales in the Genome Taxonomy Database (GTDB; R05-RS95 17 July 2020) [15]. Twenty-one genomes within the family Christensenellaceae are included in the GTDB R05-RS95 as of 1 August 2020. These include metagenomeassembled genomes and genomes derived from isolates. A formal reclassification of Catabacter as Christensenella would clarify the nomenclature of this taxon.
Here, we used comparative genomics as a basis for proposing the transfer of Catabacter hongkongensis to the genus of Christensenella. Genome sequences of six cultured isolates belonging to the families 'Catabacteraceae' and Christensenellaceae and four species from sister clades in the GTDB were OPEN ACCESS selected for phylogenomic analysis. The average nucleotide identity (ANI) of the six genomes was compared, and a phylogeny based on 16S rRNA gene sequences was reconstructed. Based on the resulting phylogeny, we recommend that Catabacter hongkongensis be renamed Christensenella hongkongensis comb. nov.
We used Anvi' o version 5.2.0 for reconstructing the wholegenome phylogenomic tree [16]. Briefly, contig databases were created from the genome fasta files. Prodigal version 2.6.3 with default settings [17] was used to identify open reading frames in contigs. Hidden Markov model (HMM) profiles were used to extract the set of single-copy marker genes defined by Campbell et al. [18] . The best HMM hit was selected if a gene was found with multiple copies in a genome. We limited the set of single-copy core genes shared to those present in all analysed genomes and aligned the concatenated protein sequences using muscle [19]. FastTree 2 [20] was used for reconstructing an approximately maximum-likelihood phylogenomic tree with the Jones-Taylor-Thornton model [21]. SH-like local support values [22] are shown on the nodes. 16S rRNA gene sequences were retrieved from NCBI and aligned using mafft. The tree was reconstructed using the maximum-likelihood method by RAxML [23] with a general time reversible model of evolution. The phylogenetic tree was visualized using the online tool iTOL [24].

Average nucleotide identity and phenotype predictions
We used FastANI with default settings [25] to generate a pairwise ANI comparison of the six Christensenella and Catabacter genomes. A heatmap of ANI values was generated and visualized in R [26] with the package ggplot2 [27]. Traitar [28] trait analyzer was used for phenotypic trait prediction based on genome sequences. ABRicate version 1.0.1 (https:// github. com/ tseemann/ ABRicate) was used for the detection of genes involved in antimicrobial resistance (AMR), and the annotation was derived from the default NCBI database AMRFinderPlus.

RESULTS AND DISCUSSION
The genome sizes of the six Catabacter and Christensenella species/strains range from 2.5 Mbp to 3.3 Mbp and the G+C content of genomic DNA from 48.53 to 52.07 mol%. Based on the pairwise comparison of the six genomes in the families 'Catabacteraceae' and Christensenellaceae, we observed that the ANI values of the two Catabacter hongkongensis strains (HKU16 T and ABBA15k) were >98.97 % (Fig. 1), confirming that the two strains belong to the same species. Moreover, the ANI values for the six genomes were between 77.56-83.48 %, which corresponds to the accepted ANI cut-off 95-96 % used to designate the same species [29,30] and <83 % for interspecies ANI values [25]. 'Christensenella intestinihominis' AF73-05CM02 PP and C. minuta DSM 22607 T showed the highest ANI similarity values (83.48 %) between different species.
The16S rRNA gene phylogeny shows Catabacter is nested within the Christensenella clade with 100 % bootstrap support (Fig. 2). The two strains of Catabacter (C. hongkongensis HKU16 T and ABBA15k) have identical 16S rRNA gene sequences. The 16S rRNA gene sequence identities between Catabacter hongkongensis and Christensenella species were between 96-97 %. Both 16S rRNA gene sequence similarity and 16S rRNA gene-based phylogenetic relationships of these taxa support that Catabacter and Christensenella belong to the same genus.
We identified 135 protein-encoding single-copy core genes present in the genomes of Christensenella, Catabacter and the outgroup taxa. We used these 135 genes in a concatenated alignment resulting in a total of 51 813 aligned amino acid sites. In the resulting phylogenetic tree (Fig. 3), the Catabacter and Christensenella species and strains formed a monophyletic clade with high bootstrap support, indicating a shared common ancestor. The species 'C. timonensis' Marseille-P2437 is basal and forms a sister clade to the rest of the taxa in the phylogeny. The two strains of Catabacter hongkongensis (HKU16 T and ABBA15k) are, as expected based on their high ANI, on the same branch of the phylogeny. The Catabacter branch is a sister taxon to the remaining Christensenella species (C. minuta DSM 22607 T , 'C. massiliensis' Marseille-P2438, 'C. intestinihominis' AF73-05CM02 PP ). The position of Catabacter (and its family 'Catabacteraceae'), nested within the Christensenella clade, splits the Christensenellaceae family and genus, such that neither are monophyletic. For the family and genus names to represent monophyletic groups, the renaming of Catabacter hongkongensis to Christensenella hongkongensis would be required. As a consequence, the genus name Catabacter should be reclassified as Christensenella.
The cultured strains of the species of Catabacter (C. hongkongensis HKU16 T and ABBA15k) and Christensenella (C. minuta DSM 22607 T , 'C. massiliensis' Marseille-P2438, 'C. timonensis' Marseille-P2437 and 'C. intestinihominis' AF73-05CM02 PP ) have been shown to be strictly anaerobic and non-spore-forming rods with varied motility, Gram stain reaction and the catalase reaction [1,[7][8][9][10]. The different phenotypic characteristics of the species compared in this study are summarized in Table 1. Catabacter hongkongensis HKU16 T and ABBA15k strains are reported to be Gram-positive, while the four species of Christensenella are reported as either Gram-positive or Gram-negative. Morotomi and colleagues reported that C. minuta DSM 22607 T is Gram-negative [7], while Alonso and colleagues reported C. minuta stains consistently as Gram-positive [31]. Based on our Gram staining, C. minuta cell membranes also stained as Gram-positive, which is consistent with the observation of Alonso and colleagues. Moreover, the phenotype predictions obtained from Traitar indicate these taxa should stain Gram-positive. The Gram-variable reaction might be due to the age of the culture for staining [32].
C. hongkongensis strains (HKU16 T , HKU17, CA1, CA2) and most clinical-derived isolates are reported to be motile and resistant to cefotaxime [1,2,5,33] except for C. hongkongensis ABBA15k, which was isolated in 2016 from the blood of a patient with a fever in Sweden [34]. Strain ABBA15k showed 100 % pairwise 16S rRNA gene identity with Catabacter hongkongensis HKU16 T . However, the genome of C. hongkongensis ABBA15k is smaller than C. hongkongensis HKU16 T , and the genes coding for chemotaxin (cheA) and flagellar assembly (flhA and MotA) were not present in the genome of C. hongkongensis ABBA15k [34]. The tetracycline resistance gene tet was detected in the genome of C. hongkongensis HKU16T, but no resistance genes were detected in the genome of C. hongkongensis ABBA15k [34].
Screening for AMR genes of the genomes with ABRicate in this study showed that the tet gene was also present in the genomes of Christensenella minuta DSM 22607 T , 'Christensenella massiliensis' Marseille-P2438, 'Christensenella timonensis' Marseille-P2437 and Catabacter hongkongensis HKU16 T but not in 'Christensenella intestinihominis' AF73-05CM02 PP and Catabacter hongkongensis ABBA15k. A streptomycin resistance gene (aadE) was also detected in the genome of 'Christensenella massiliensis' Marseille-P2438. Detailed information about AMR genes is listed in Table 2. 'Christensenella intestinihominis' AF73-05CM02 PP and Catabacter hongkongensis HKU16 T were predicted to be motile by Traitar. However, 'Christensenella intestinihominis' AF73-05CM02 PP was classified as non-motile in the original phenotypic characterization [10], which might be attributable to the growth conditions used. It is also possible that the genome of the strain may not contain all genes required for flagellar formation.
In conclusion, both Catabacter and Christensenella include species and strains that are strictly anaerobic, non-spore forming, short straight rods and have diverse phenotypes regarding motility, Gram-staining and antibiotic resistance. The description of Christensenella hongkongensis is identical to that proposed for Catabacter hongkongensis [1].

Funding information
This work was supported by the Max Planck Society.