Genome-Based Taxonomy of Brevundimonas with Reporting Brevundimonas huaxiensis sp. nov

ABSTRACT Brevundimonas is a genus of Gram-negative bacteria widely distributed in nature and is also an opportunistic pathogen causing health care-associated infections. Brevundimonas strain 090558T was recovered from a blood culture of a cancer patient and was subjected to genome sequencing and analysis. The average nucleotide identity and in silico DNA-DNA hybridization values between 090558T and type strains of Brevundimonas species were 78.76% to 93.94% and 19.8% to 53.9%, respectively, below the cutoff to define bacterial species. Detailed phenotypic tests were performed, suggesting that 090558T can be differentiated from other Brevundimonas species by its ability to assimilate sodium acetate but not to utilize glucose, trypsin, or β-glucosidase. Strain 090558T (GDMCC 1.1871T or KCTC 82165T) therefore represents a novel Brevundimonas species, for which the name Brevundimonas huaxiensis sp. nov. is proposed. All Brevundimonas genomes available in GenBank (accessed on 25 January 2021) were retrieved, discarding those labeled “excluded from RefSeq” by GenBank, and included 82 genomes for precise species curation. In addition to the 21 Brevundimonas species with genomes of type strains available, we identified 29 Brevundimonas taxa that either belong to the 12 Brevundimonas species without available genomes of type strains or represent novel species. We found that more than half (57.3%) of the 82 Brevundimonas genomes need to be corrected for species assignation, including species mislabeling of a type strain. Our analysis highlights the complexity of Brevundimonas taxonomy. We also found that only some Brevundimonas species are associated with human infections, and more studies are warranted to understand their pathogenicity and epidemiology. IMPORTANCE Brevundimonas is a genus of the family Caulobacteraceae and comprises 33 species. Brevundimonas can cause various infections but remains poorly studied. In this study, we reported a novel Brevundimonas species, Brevundimonas huaxiensis, based on genome and phenotype studies of strain 090558T recovered from human blood. We then examined the species assignations of all Brevundimonas genomes (n = 82) in GenBank and found that in addition to the known Brevundimonas species with genome sequences of type strains available, there are 29 Brevundimonas taxa based on genome analysis, which need to be further studied using phenotype-based methods to establish their species status. Our study significantly updates the taxonomy of Brevundimonas and enhances our understanding of this genus of clinical relevance. The findings also encourage future studies on the characterization of novel Brevundimonas species.

T he genus Brevundimonas was first proposed by Segers et al. (1). Brevundimonas strains are Gram-negative, motile bacteria of the family Caulobacteraceae; contain Q-10 as the major isoprenoid quinone; and have high DNA G1C contents (2)(3)(4). At the time of writing, the genus Brevundimonas comprises 33 species (Table 1). Brevundimonas can be found in diverse environments such as soils (5), water (3,6,7), activated sludge (8), and plant roots (9). Brevundimonas has the potential to be used for a wide range of activities such as cadmium biosorption (10), soil bioremediation (11), water pollutant treatment (12), and plant growth promotion for sustainable agriculture in arid regions (13). In humans, Brevundimonas is an opportunistic pathogen able to cause a range of hospital-acquired infections such as bacteremia, eye infection, peritonitis, urinary tract infection, and skin and soft tissue infection (14). In this study, we report a clinical strain from blood to represent a novel Brevundimonas species, Brevundimonas huaxiensis, based on genome and phenotype studies. We also curated all Brevundimonas genomes available in GenBank for precise species identification and found at least 17 tentative novel Brevundimonas species.

RESULTS
Strain 090558 T represents a novel Brevundimonas species with the proposed name Brevundimonas huaxiensis. Strain 090558 T was recovered from a blood culture of a 46-year-old male patient with hepatocellular carcinoma. The patient received liver surgery 18 days prior to the collection of the blood culture but had no central lines, drainage tubes, or any other invasive devices within 2 weeks before the blood culture. The source of this strain is unknown. The strain was preliminarily identified as Brevundimonas  (Table 1). However, strain 090558 T appears to be well separated from other Brevundimonas species in the phylogenetic tree based on the 16S rRNA gene sequence (see Fig. 1 for the maximum likelihood tree; the neighbor-joining and maximum parsimony trees are shown in Fig. S1 and S2 in the supplemental material). It is well known that analysis based on 16S rRNA gene sequences is not sufficient for precise species identification of bacterial species (15). We therefore performed whole-genome sequencing using a short-read Illumina sequencer for strain 090558 T .
A total of 6,478,361 reads and 1.94 GB of clean bases were generated, which were then assembled into 35 contigs (N 50 , 355,886 bp). The draft genome of strain 090558 T is 3,163,842 bp in size with a 66.4 mol% G1C content. JCM 11415 T , the type strain of B. nasdae, had .99% 16S rRNA sequence identity with strain 090558 T (see below) but had no genome sequences or sequences of housekeeping genes for comparison. We therefore obtained B. nasdae type strain JCM 11415 T and also sequenced the strain using HiSeq X10, which generated a total of 5,993,515 reads and 1.80 GB of clean bases, which were then assembled into 39 contigs (N 50 , 260,309 bp). The draft genome of strain JCM 11415 T is 3,751,817 bp in size with a 65.6 mol% G1C content.
In the maximum likelihood phylogenomic tree (Fig. 2) based on the 1,040 core genes of all Brevundimonas genomes available in GenBank (see Data Set S1 in the supplemental  Table 1). The tree was inferred using the maximum likelihood method. Caulobacter mirabilis FWC 38 T (GenBank accession no. CP024201) was used as an outgroup. Bootstrap values of .70% (based on 1,000 resamplings) are shown. Bar, 0.01 substitutions per nucleotide position.

FIG 2
Phylogenomic tree based on the concatenated nucleotide sequences of core genes of strain 090558 T and all Brevundimonas genomes available in GenBank (accessed on 25 January 2021) (see Data Set S1 in the supplemental material). Strains and their nucleotide accession numbers are listed alongside the labeled species names in GenBank and precise species names assigned in this study. The tree was inferred using the maximum likelihood method under a GTR gamma model with a 1,000-bootstrap test, and branches with support of over 50% are indicated by gradients. The bar indicates nucleotide substitutions per site. material), strain 090558 T was well situated within the genus Brevundimonas and was most closely related to B. vesicularis among known Brevundimonas species. The average nucleotide identity (ANI) values between strain 090558 T and the type strains of all Brevundimonas species with genome sequences ranged from 78.76% to 93.94%, lower than the $95 to 96% cutoff for defining species (16) ( Table 2). Consistently, the in silico DNA-DNA hybridization (isDDH) values between strain 090558 T and the type strains of all Brevundimonas species with genome sequences ranged from 19.8% to 53.9% (Table 2), which are below the $70.0% cutoff to define species (16,17). The optimal isDDH cutoff to define a bacterial genus has not been established, and relatively low (around 20%) isDDH values between species within the same genus have been reported previously, such as for Acinetobacter (18) and Pseudomonas (19). Of note, among the five species having .98.5% 16S rRNA gene sequence identity with strain 090558 T , the type strain of B. intermedia has no available genome sequences. Nonetheless, the housekeeping gene gyrB (encoding DNA gyrase subunit B) of strain 090558 T had only 82.19% nucleotide identity to that of the B. intermedia CIP 106444 T (GenBank accession no. EU024185). This suggests that strain 090558 T does not belong to B. intermedia.
The above-described analyses clearly suggest that strain 090558 T represents a novel species of the genus Brevundimonas. As the strain was recovered from West China Hospital, we propose the species name Brevundimonas huaxiensis (hua.xi.en9sis. fem. adj. huaxiensis, referring to West China [huaxi in Chinese], where the type strain was recovered) after phenotypic characterization (see below). Strain 090558 T has been deposited into the Guangdong Microbial Culture Collection Center as GDMCC1.1871 T and into the Korean Collection for Type Cultures as KCTC 82165 T .
B. huaxiensis can be differentiated from other Brevundimonas species by its ability to assimilate sodium acetate but not to utilize glucose, trypsin, or b-glucosidase. Cells of strain 090558 T were Gram stain negative, aerobic, motile, non-spore forming, and rod shaped (0.3 to 0.5 mm in diameter and 1.0 to 2.5 mm long) (Fig. S3). Strain 090558 T grew on nutrient agar, tryptic soy agar, R2A agar, MacConkey agar, and brain heart infusion (BHI) agar after 2 days. Colonies grown on nutrient agar at 35°C after 2 days were circular, smooth, convex, and orange. Growth occurs at 8°C to 42°C in the presence of 0 to 4% (wt/vol) NaCl and at pH 6.0 to 8.0. It is negative for oxidase and activities of arylamidase, arginine dihydrolase, cystine, a-chymotrypsin, a-fucosidase, a-ga- acetyl-b-glucosaminidase, and trypsin. Catalase, PNPG (p-nitrophenyl-b-D-galactopyranoside), indole production, and nitrate reduction tests and activities of acid phosphatase, alkaline phosphatase, esterase (C 4 ), esterase lipase (C 8 ), a-glucosidase, leucine arylamidase, naphthol-AS-BI(7-bromo-3-hydroxy-2-naphtho-o-anisidine)-phosphohydrolase, urease, and valine arylamidase are positive. Tween 40, esculin, and gelatin are hydrolyzed, but starch, DNase, and cellulose are not. It is able to assimilate N-acetylglucosamine, potassium gluconate, and sodium acetate but does not utilize adipic acid, arabinose, capric acid, glucose, malic acid, maltose, mannose, mannitol, phenylacetic acid, and trisodium citrate. It can assimilate sodium acetate instead of glucose. It is negative for oxidase and activities of trypsin and b-glucosidase. The ability to assimilate sodium acetate but not to utilize glucose, trypsin, or b-glucosidase can differentiate 090558 T from other Brevundimonas species (Table 3). The isoprenoid quinone of strain 090558 T was Q-10, which is typical of members of the genus Brevundimonas. The major cellular fatty acids were summed feature 8 (comprising C 18:1 v 7c/C 18:1 v 6c) (55.87%) and C 16:0 (25.2%), which are the same as those of closely related Brevundimonas species (Table S1).
Curation of Brevundimonas genomes available in GenBank. With the inclusion of B. huaxiensis identified in this study, there are 34 known Brevundimonas species at present (Table 4). Genome sequences of type strains are available for 22 Brevundimonas species, including B. huaxiensis 090558 T and B. nasdae JCM 11415 T sequenced in this study. We then determined pairwise ANI and isDDH values between the type strain genomes. We found that the genome of Brevundimonas denitrificans strain TAR-002 T (GenBank accession no. BEWU00000000) had 99.78% ANI and 99.1% isDDH with that of Brevundimonas abyssalis TAR-001 T (GenBank accession no. BATC00000000) (Data Set S2), suggesting that the two  strains actually belonged to the same species or that the strains were mislabeled. We therefore checked the identity of the two genomes with their corresponding 16S rRNA gene sequences available in GenBank and found only 93.97% identity between the genome of TAR-002 T and its original 16S rRNA sequence (GenBank accession no. AB899817). It is therefore likely that the genome of TAR-002 T was mislabeled for another strain of B. abyssalis. Genome sequences of type strains of the remaining 20 species have ,95% ANI and ,70% isDDH values between each other (Data Set S2). There were 60 genome sequences of Brevundimonas non-type strains that were also available in GenBank. Among the 60 genomes, based on ANI and isDDH analyses with type strains of Brevundimonas species, 24 could be assigned to a known Brevundimonas species, while 36 could not (Data Set S1). Instead, a total of 29 taxa, which do not belong to any of the 21 Brevundimonas species with genome sequences of type strains available, could be identified from the 36 genomes and are assigned as taxa 1 to 29 here (Data Set S1). The closest species of the 29 taxa are listed in Table 5. More than half (n = 47 [57.3%; 47/82]) of the 82 available Brevundimonas genomes need to be corrected for species assignation. It is suggested based on the genome comparison reported here that (i) 15 genomes labeled with a Brevundimonas species name should be assigned to another Brevundimonas species (the above-mentioned strain TAR-002) or a proposed Brevundimonas taxon (the remaining 14), (ii) 7 genomes labeled as Brevundimonas sp. should be assigned to a known Brevundimonas species, and (iii) the remaining 25 genomes labeled as Brevundimonas sp. could be classified into 20 proposed taxa. Virulence factors of Brevundimonas. As strain 090558 T was recovered from a human blood culture and Brevundimonas is known as an opportunistic pathogen (14), we attempted to identify potential virulence factors of 090558 T and other Brevundimonas species. However, there is a lack of studies of Brevundimonas pathogenicity. We therefore used the Virulence Factor Database (VFDB) to predict potential virulence factors for all of the 21 Brevundimonas species with genome sequences of type strains available, including B. huaxiensis. Four potential virulence factors in the VFDB were identified in B. huaxiensis, i.e., acpXL (encoding acyl carrier protein of lipopolysaccharide [LPS]), bvrR (encoding the transcriptional regulatory protein BvrR), icl (encoding an isocitrate lyase), and a gene named Rv0440 (encoding the chaperonin GroEL) (Data Set S3). acpXL is also present in all other Brevundimonas species, while bvrR is absent only from Brevundimonas diminuta, and icl is absent only from Brevundimonas lutea (Data Set S3). Rv0440 is present in seven other Brevundimonas species (Brevundimonas aveniformis, Brevundimonas basaltis, Brevundimonas denitrificans, Brevundimonas fluminis, Brevundimonas halotolerans, Brevundimonas lenta, and Brevundimonas viscosa) (Data Set S3). No additional virulence factors were identified in the two major Brevundimonas species causing human infections, B. diminuta and B. vesicularis (14).

DISCUSSION
In this study, we report a novel Brevundimonas species, B. huaxiensis, based on both phenotypic and genomic analyses. We identified that the genome of B. denitrificans strain TAR-002 T actually belonged to another strain of B. abyssalis. We then applied the updated taxonomic assignations to curate genome sequences deposited in GenBank with the label of Brevundimonas and found the presence of 29 taxa, which were different from the 21 known Brevundimonas species with available genome sequences of type strains. We also found that the species identification of more than half of the Brevundimonas genome sequences in GenBank needs to be corrected. The above-described findings suggest the complicated taxonomy of Brevundimonas and the need for careful curation of genomes labeled as Brevundimonas.
For the 29 taxa identified among Brevundimonas genomes in GenBank in this study, it is possible that some are actually one of the 12 known Brevundimonas species but with no genome sequences of type strains being available. However, most of the 29 taxa (at least 17 taxa) appear to actually represent novel Brevundimonas species, which are unnamed as they have not been characterized by phenotype methods and therefore warrant further studies.
Only half of all Brevundimonas genomes in GenBank have information on the isolation source available (see Data Set S1 in the supplemental material). Among the 7 Brevundimonas genomes that are labeled with humans as the source, 3 belonged to B. diminuta, and 1 each belonged to taxon 2 closest to Brevundimonas naejangsanensis ([ Table 5]), taxon 17 (closest to B. diminuta [ Table 5]), B. vesicularis, and B. huaxiensis (090558 T ). B. diminuta and B. vesicularis are known as the two major Brevundimonas species causing human infections (14). However, species identification in clinical microbiology laboratories is commonly based on phenotypic features, but it is well known that such phenotype-based approaches can cause misidentification and are unreliable for precise species identification(e.g., see references [20][21][22]. Therefore, B. diminuta and B. vesicularis reported in clinical cases may actually belong to other Brevundimonas species. Even the genome label in the NCBI database may not be correct or needs to be updated. For instance, two genomes of Brevundimonas strains recovered from humans labeled as B. diminuta in GenBank actually belong to taxa 2 and 17, respectively (Data Set S1). Nonetheless, it appears that among the 34 Brevundimonas species, few are associated with human infections, including B. diminuta (23), B. vesicularis (24), B. vancanneytii (25), and B. huaxiensis (this study). The pathogenicities of these Brevundimonas species have not been characterized (14), and the prediction of virulence factors using the VFDB for Brevundimonas species is preliminary. The virulence of Brevundimonas warrants further studies.
We are aware of the limitations of this study. First, the phenotypic features and the fatty acid contents of strain 090558 T were compared with those of other Brevundimonas species reported in references rather than, ideally, in the same experiments. Second, as mentioned above, there are no genome sequences available for type strains of 12 known Brevundimonas species, although they are not closely related to strain 090558 T . Third, there is only a single strain for the novel species identified here. Despite these limitations, the novel species status of B. huaxiensis can be established by the above-mentioned detailed analyses.
In conclusion, we identified and characterized a novel Brevundimonas species, B. huaxiensis, which is associated with human infection and is therefore of clinical relevance. B. huaxiensis can be differentiated from other Brevundimonas species by its ability to assimilate sodium acetate but not to utilize glucose, trypsin, or b-glucosidase. The genus Brevundimonas has 34 known species and at least 17 potential novel, unnamed species. Currently, few Brevundimonas species have been found to be associated with human infections. Genome sequencing of type strains of all Brevundimonas species is required to further untangle the taxonomic complexity of the genus. The proposed Brevundimonas taxa warrant further characterization using both phenotype-and genome-based approaches to establish their proper species assignations. More studies of the human-associated Brevundimonas species are required to understand their pathogenicity and epidemiology in clinical infections.

MATERIALS AND METHODS
Strain. Strain 090558 T was recovered from the blood of a 46-year-old patient at West China Hospital of Sichuan University, Chengdu, China, in November 2019. Preliminary species identification was performed using Vitek II (bioMérieux, Marcy l'Etoile, France).
Analysis of the 16S rRNA gene sequence. Genomic DNA of strain 090558 T was prepared using a bacterial DNA kit (Tiangen, Beijing, China). The 16S rRNA gene was amplified by PCR using the universal primers 27F and 1492R (26) and Sanger sequencing for preliminary species identification. 16S rRNA gene sequences of related species were retrieved from GenBank and aligned with that of strain 090558 T using Clustal Omega (27). A maximum likelihood tree was inferred in RAxML v8.2.12 (28), and two more trees were inferred based on the neighbor-joining and maximum parsimony algorithms using MEGA v10.2.6 (29) with a 1,000-bootstrap test.
Whole-genome sequencing and analysis. Whole-genome sequencing of strain 090558 T was performed on the Illumina HiSeq X10 platform (Illumina, San Diego, CA, USA). JCM 11415 T , the type strain of B. nasdae, had .99% 16S rRNA sequence identity with strain 090558 T but had no genome sequences or sequences of housekeeping genes for comparison. We therefore obtained B. nasdae strain JCM 11415 T from the Japan Collection of Microorganisms (JCM) via Shanghai Yansheng Ltd. and also sequenced the strain using the HiSeq X10 platform. Reads were de novo assembled into contigs using SPAdes v3.13.0 (30), applying the careful and auto-cutoff modes. The draft genome of strain 090558 T was compared with those of type strains of Brevundimonas species using the average nucleotide identity (ANI) based on BLAST analysis and in silico DNA-DNA hybridization (isDDH). ANI and isDDH values were calculated using JSpecies (16) and Genome-to-Genome Distance Calculator (formula 2) (17) with the recommended parameters and/or default settings, respectively. A $70.0% isDDH value (16,17) and a $95 to 96% ANI value (16) were used as the cutoffs to define a bacterial species.
Phenotypic characterization and in vitro susceptibility testing. Growth on nutrient agar, tryptic soy agar, R2A agar, MacConkey agar, and blood heart infusion (BHI) agar (all from Hopebio, Qingdao, China) was examined at 35°C for 2 days. Cell motility was examined by observing bacterial growth and diffusion on a deep semisolid nutrient agar medium of 0.3% (wt/vol) agar (Hopebio). Anaerobic growth was examined by streaking the bacterial cultures on brain heart infusion agar plates and placing them in a GasPak EZ anaerobic bag (BD, Franklin Lakes, NJ, USA) at 30°C for 5 days. After incubation in R2A broth at 30°C for 3 days, flagella of strain 090558 T were observed with an H-7650 transmission electron microscope (Hitachi, Tokyo, Japan). The growth of strain 090558 T was examined in 5-ml aliquots of R2A broth dispensed into tubes (16-mm inner diameter) at temperatures of 4°C, 8°C, 18°C, 28°C, 32°C, 37°C, 42°C, 45°C, 48°C, and 50°C. Salt and pH tolerances were measured using R2A broth at 30°C for 5 days at different NaCl concentrations (0.5, 1, 2, 3, 4, 5, 7.5, 10, and 15% [wt/vol]) and various pHs (pH 4.0 to 12.0, in increments of 1.0 unit), respectively. A catalase activity test was conducted by examining the production of bubbles after the addition of a 3% (vol/vol) hydrogen peroxide solution, while oxidase activity was tested by using a 1% tetramethyl-p-phenylenediamine dihydrochloride solution. DNase activity was detected with 1 M HCl using DNase agar (Solarbio, Beijing, China) after 3 days of incubation at 30°C. Malonate, phenylalanine deaminase, and potassium cyanide (KCN) experiments were performed using biochemical identification tubes (Huankai, Guangzhou, Guangdong, China). Commercially available API 20NE and API Zym kits (bioMérieux) were used for testing biochemical features and enzyme activities.
Fatty acid analysis. The analysis of cellular fatty acids, quinones, and polar lipids was performed by the Guangdong Institute of Microbiology (Guangzhou, Guangdong). Briefly, fatty acid methyl esters were extracted and analyzed by gas chromatography according to the instructions of the Sherlock microbial identification system (MIDI Inc., Newark, DE, USA) as described previously (35,36). Peaks were automatically integrated, and fatty acid proportions were calculated using the MIDI identification database RTSBA6 (version 6.00; MIDI Inc.).
Virulence factor prediction. Type strains of the genus Brevundimonas with genomes available (n = 20) as well as B. huaxiensis 090558 T and B. nasdae JCM 11415 T sequenced in this study were screened for the presence of virulence factors against 32,838 sequences (accessed on 6 March 2021) retrieved from data set B in the Virulence Factor Database (VFDB) (37) by a nucleotide similarity search using local BLAST (38). Hits with either coverage or identity of below 70% were discarded in the results.
Curation of species identification for Brevundimonas genomes in GenBank. We used txid41275 [Organism:exp] AND "latest" [filter] to search NCBI GenBank, and a total of 175 assemblies were available (accessed on 25 January 2021) in the genus Brevundimonas. We discarded 93 genomes that were labeled as "excluded from RefSeq" by NCBI GenBank due to either (i) being derived from a metagenome, (ii) having a fragmented assembly, (iii) having a genome length too small, or (iv) having many frameshifted proteins. Therefore, 82 Brevundimonas genome sequences (type strains of 22 species and 60 non-type strains) were included (Data Set S1), all of which were retrieved and then subjected to precise species identification using ANI and isDDH analyses as described above. Strains that have both a ,70% isDDH value and a ,96% ANI value with any known Brevundimonas species are likely to belong to a novel species, which is temporarily assigned a taxon here as the establishment of a novel species requires phenotypic characterization in addition to genome analysis.
Data availability. The draft genome sequence and the nearly complete 16S rRNA gene of strain 090558 T have been deposited in the DDBJ/EMBL/GenBank database under accession no. JABBJE000000000