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

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.

INTRODUCTION

The 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 G+C contents (2–4). 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.

TABLE 1

Nucleotide identities of 16S rRNA gene sequences between strain 090558T and type strains of Brevundimonas species

Species	Strain	GenBank accession no.	Identity (%)
B. vesicularis	NBRC 12165T	AB680247	99.71
B. nasdae	JCM 11415T	AB071954	99.64
B. intermedia	ATCC 15262T	AJ227786	99.49
B. aurantiaca	DSM 4731T	AJ227787	99.13
B. mediterranea	V4.BO.10T	AJ227801	98.91
B. olei	MJ15T	GQ250440	98.34
B. albigilva	NHI-13T	KC733808	98.27
B. naejangsanensis	DSM 23858T	ATXN01000003	97.98
B. kwangchunensis	KSL-102T	AY971368	97.76
B. viscosa	CGMCC 1.10683T	jgi.1076140	97.69
B. humi	CA-15T	KY117472	97.61
B. faecalis	CS20.3T	FR775448	97.58
B. alba	DSM 4736T	AJ227785	97.40
B. bacteroides	DSM 4726T	JNIX01000007	97.39
B. poindexterae	FWC40T	AJ227797	97.18
B. halotolerans	MCS24T	QTTA01000016	97.18
B. diminuta	ATCC 11568T	GL883089	97.18
B. bullata	IAM 13153T	D12785	97.18
B. vancanneytii	LMG 2337T	AJ227779	97.11
B. balnearis	FDRGB2bT	LN651199	97.11
B. staleyi	FWC43T	AJ227798	97.11
B. lenta	DS-18T	EF363713	96.97
B. terrae	KSL-145T	DQ335215	96.89
B. denitrificans	TAR-002T	AB899817	96.89
B. subvibrioides	ATCC 15264T	ADBM01000034	96.89
B. fluminis	LA-55T	RQWJ01000003	96.82
B. variabilis	ATCC 15255T	AJ227783	96.75
B. basaltis	J22T	EU143355	96.64
B. mongoliensis	R-10-10T	MF436701	96.61
B. aveniformis	DSM 17977T	AUAO01000001	96.58
B. lutea	NS26T	KX601076	95.12
B. abyssalis	TAR-001T	BATC01000012	94.88
B. canariensis	GTAE24T	KX898252	94.73

Type strains having >98.5% 16S rRNA gene sequence identity with strain 090558T are highlighted in boldface type.

RESULTS

Strain 090558T represents a novel Brevundimonas species with the proposed name Brevundimonas huaxiensis.

Strain 090558T 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 vesicularis by Vitek II (bioMérieux, Marcy l’Etoile, France). The strain was susceptible to amikacin, ampicillin, ampicillin-sulbactam, aztreonam, ceftriaxone, ceftazidime, cefepime, cefotaxime, cefuroxime, chloramphenicol, ciprofloxacin, colistin, imipenem, meropenem, piperacillin-tazobactam, sulfamethoxazole-trimethoprim, and tigecycline but resistant to aztreonam. The patient responded well to treatment with cefoperazone-sulbactam and was discharged from the hospital later.

The nearly complete 16S rRNA gene sequence of strain 090558T (1,383 bp) was obtained, which had the highest identity to those of Brevundimonas vesicularis NBRC 12165T (99.71%), Brevundimonas nasdae JCM 11415T (99.64%), Brevundimonas intermedia ATCC 15262T (99.49%), Brevundimonas aurantiaca DSM 4731T (99.13%), and Brevundimonas mediterranea V4.BO.10T (98.91%) (Table 1). However, strain 090558T 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 090558T.

FIG 1

Phylogenetic tree based on 16S rRNA gene sequences of strain 090558T and type strains of Brevundimonas species (Table 1). The tree was inferred using the maximum likelihood method. Caulobacter mirabilis FWC 38T (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.

A total of 6,478,361 reads and 1.94 GB of clean bases were generated, which were then assembled into 35 contigs (N50, 355,886 bp). The draft genome of strain 090558T is 3,163,842 bp in size with a 66.4 mol% G+C content. JCM 11415T, the type strain of B. nasdae, had >99% 16S rRNA sequence identity with strain 090558T (see below) but had no genome sequences or sequences of housekeeping genes for comparison. We therefore obtained B. nasdae type strain JCM 11415T 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 (N50, 260,309 bp). The draft genome of strain JCM 11415T is 3,751,817 bp in size with a 65.6 mol% G+C 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 material), strain 090558T 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 090558T 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 090558T 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 090558T, the type strain of B. intermedia has no available genome sequences. Nonetheless, the housekeeping gene gyrB (encoding DNA gyrase subunit B) of strain 090558T had only 82.19% nucleotide identity to that of the B. intermedia CIP 106444T (GenBank accession no. EU024185). This suggests that strain 090558T does not belong to B. intermedia.

FIG 2

Phylogenomic tree based on the concatenated nucleotide sequences of core genes of strain 090558T 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.

TABLE 2

ANI and isDDH values between 090558T and type strains of Brevundimonas species

Species	Strain	GenBank accession no.	ANI (%)	isDDH value (%)
B. abyssalis	TAR-001T	BATC00000000	80.28	20.30
B. alba	DSM 4736T	JAATJM000000000	81.08	21.00
B. aurantiaca	DSM 4731T	JACHOQ000000000	85.40	26.40
B. aveniformis	DSM 17977T	AUAO00000000	78.76	20.80
B. bacteroides	DSM 4726T	JNIX00000000	80.70	20.60
B. basaltis	DSM 25335T	JACHFZ000000000	80.92	20.90
B. bullata	HAMBI 262T	QLLC00000000	82.65	21.80
B. diminuta	ATCC 11568T	GL883089	82.46	22.10
B. fluminis	LA-55T	RQWJ00000000	80.58	20.50
B. halotolerans	MCS24T	QTTA01000000	80.47	20.20
B. lenta	DSM 23960T	JACIDM000000000	81.74	21.70
B. lutea	NS26T	QUOQ00000000	80.21	20.80
B. mediterranea	DSM 14878T	JACIDA000000000	86.16	27.60
B. naejangsanensis	DSM 23858T	ATXN00000000	82.13	21.30
B. nasdae	JCM 11415T	JAEPWN000000000	85.25	25.80
B. subvibrioides	ATCC 15264T	ADBM00000000	81.19	21.00
B. terrae	DSM 17329T	JAASQT000000000	78.98	19.80
B. variabilis	DSM 4737T	JACHOR000000000	79.84	20.10
B. vesicularis	NBRC 12165T	BCWM00000000	93.94	53.90
B. viscosa	CGMCC 1.10683T	FOZV00000000	80.83	20.70

Type strains having >98.5% 16S rRNA gene sequence identity with strain 090558T (Table 1) are highlighted in boldface type.

The above-described analyses clearly suggest that strain 090558T 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.en′sis. fem. adj. huaxiensis, referring to West China [huaxi in Chinese], where the type strain was recovered) after phenotypic characterization (see below). Strain 090558T has been deposited into the Guangdong Microbial Culture Collection Center as GDMCC1.1871T and into the Korean Collection for Type Cultures as KCTC 82165T.

B. huaxiensis can be differentiated from other Brevundimonas species by its ability to assimilate sodium acetate but not to utilize glucose, trypsin, or β-glucosidase.

Cells of strain 090558T were Gram stain negative, aerobic, motile, non-spore forming, and rod shaped (0.3 to 0.5 μm in diameter and 1.0 to 2.5 μm long) (Fig. S3). Strain 090558T 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, α-chymotrypsin, α-fucosidase, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, lipase (C14), α-mannosidase, N-acetyl-β-glucosaminidase, and trypsin. Catalase, PNPG (p-nitrophenyl-β-d-galactopyranoside), indole production, and nitrate reduction tests and activities of acid phosphatase, alkaline phosphatase, esterase (C4), esterase lipase (C8), α-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 β-glucosidase. The ability to assimilate sodium acetate but not to utilize glucose, trypsin, or β-glucosidase can differentiate 090558T from other Brevundimonas species (Table 3).

TABLE 3

Phenotypic characteristics of strain 090558T and closely related strains of the genus Brevundimonas

Characteristic	Result or value for strain
	090558T	B. aurantiaca	B. vesicularis	B. intermedia	B. nasdae	B. mediterranea
Colony color	Orange	Yellow	Orange	Cream	Slightly yellow	Cream white
Property
Motility	+	+	+	+	+	+
Esculin hydrolysis	+	+	+	+	+	−
PNPG	+	−	+	−	+	−
Assimilation of:
Glucose	−	+	+	−	+	−
Mannose	−	−	−	−	−	−
N-Acetylglucosamine	+	−	W	−	+	−
Maltose	−	W	+	−	+	−
Malic acid	−	W	+	−	+	+
Trisodium citrate	−	−	−	−	−	−
Phenylacetic acid	−	−	−	−	−	−
Sodium acetate	+	−	−	−	+	+
Enzyme activity
Oxidase	−	+	+	+	+	+
Catalase	+	+	+	+	+	−
Lipase (C14)	−	−	W	−	W	−
Leucine arylamidase	+	+	+	W	W	+
Valine arylamidase	+	W	W	+	+	W
Trypsin	−	+	W	W	+	+
α-Chymotrypsin	−	W	W	W	W	W
Acid phosphatase	+	+	+	+	+	+
α-Galactosidase	−	−	−	−	+	−
β-Galactosidase	−	W	+	W	W	−
β-Glucuronidase	−	−	−	−	−	−
α-Glucosidase	+	W	W	W	+	−
β-Glucosidase	−	−	+	−	+	−
G+C content (mol%)	66.4	65.6	65.0–66.0	66.1	66.5	67.3

+, positive; −, negative; W, weakly positive. Data for species other than B. huaxiensis are from reference 2. Closely related species refer to those having >98.5% 16S rRNA gene sequence identity with strain 090558T (Table 1).

The isoprenoid quinone of strain 090558T was Q-10, which is typical of members of the genus Brevundimonas. The major cellular fatty acids were summed feature 8 (comprising C18:1 ω7c/C18:1 ω6c) (55.87%) and C16:0 (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 090558T and B. nasdae JCM 11415T 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-002T (GenBank accession no. BEWU00000000) had 99.78% ANI and 99.1% isDDH with that of Brevundimonas abyssalis TAR-001T (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-002T and its original 16S rRNA sequence (GenBank accession no. AB899817). It is therefore likely that the genome of TAR-002T 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).

TABLE 4

Updated taxonomy of Brevundimonas, including 34 species at present

Species	Strain	GenBank accession no. of genome sequence	Reference
B. abyssalis	TAR-001T	BATC00000000	39
B. alba	DSM 4736T	JAATJM000000000	40
B. albigilva	NHI-13T		2
B. aurantiaca	DSM 4731T	JACHOQ000000000	40
B. aveniformis	DSM 17977T	AUAO00000000	8
B. bacteroides	DSM 4726T	AUAO00000000	40
B. balnearis	FDRGB2bT		3
B. basaltis	J22T	JACHFZ000000000	41
B. bullata	IAM 13153T	QLLC00000000	42
B. canariensis	GTAE24T		9
B. denitrificans	TAR-002T	—a	43
B. diminuta	ATCC 11568T	GL883089	1
B. faecalis	CS20.3T		44
B. fluminis	LA-55T	RQWJ00000000	6
B. halotolerans	MCS24T	QTTA00000000	45
B. huaxiensis	090558T	JABBJE000000000	This study
B. humi	CA-15T		5
B. intermedia	ATCC 15262T		40
B. kwangchunensis	KSL-102T		46
B. lenta	DS-18T	JACIDM000000000	47
B. lutea	NS26T	QUOQ00000000	7
B. mediterranea	V4.BO.10T	JACIDA000000000	4
B. mongoliensis	R-10-10T		48
B. naejangsanensis	DSM 23858T	ATXN00000000	42
B. nasdae	JCM 11415T	JAEPWN000000000	49
B. olei	MJ15T		50
B. poindexterae	FWC40T		45
B. staleyi	FWC43T		45
B. subvibrioides	ATCC 15264T	CP002102	40
B. terrae	KSL-145T	JAASQT000000000	51
B. vancanneytii	LMG 2337T		25
B. variabilis	ATCC 15255T	JACHOR000000000	40
B. vesicularis	NBRC 12165T	BCWM00000000	1
B. viscosa	CGMCC 1.10683T	FOZV00000000	52

GenBank accession no. BEWU00000000 of TAR-002T is mislabeled and actually belongs to B. abyssalis.

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.

TABLE 5

Reference strains and closest species of the Brevundimonas taxa identified based on genome sequences in this study

Taxon	Reference strain	GenBank accession no.	Closest species	ANI (%)	isDDH value (%)
1	DSM 14878	GCA_014196125.1	B. mediterranea	94.79	57.30
2	DSM 23858	GCA_000421705.1	B. naejangsanensis	94.22	54.30
3	090558	GCA_014218725.1	B. huaxiensis	95.33	63.90
4	NBRC 12165	GCA_001592205.1	B. vesicularis	95.00	59.90
5	NBRC 12165	GCA_001592205.1	B. vesicularis	95.18	60.20
6	NCTC8545	GCA_900445995.1	B. diminuta	81.94	21.20
7	DSM 23960	GCA_014196335.1	B. lenta	85.92	25.60
8	DSM 4736	GCA_011927945.1	B. alba	84.90	24.60
9	NBRC 12165	GCA_001592205.1	B. vesicularis	95.29	62.40
10	DSM 23858	GCA_000421705.1	B. naejangsanensis	93.45	49.90
11	NBRC 12165	GCA_001592205.1	B. vesicularis	95.28	61.90
12	NCTC8545	GCA_900445995.1	B. diminuta	92.94	47.70
13	ATCC 15264	GCA_000144605.1	B. subvibrioides	86.54	28.80
14	090558	GCA_014218725.1	B. huaxiensis	88.06	30.80
15	HAMBI_262	GCA_003350205.1	B. bullata	89.96	36.20
16	NBRC 12165	GCA_001592205.1	B. vesicularis	94.38	55.80
17	NCTC8545	GCA_900445995.1	B. diminuta	93.52	47.90
18	090558	GCA_014218725.1	B. huaxiensis	89.28	34.50
19	NCTC8545	GCA_900445995.1	B. diminuta	82.37	21.90
20	090558	GCA_014218725.1	B. huaxiensis	95.24	62.00
21	DSM 23960	GCA_014196335.1	B. lenta	84.19	24.20
22	090558	GCA_014218725.1	B. huaxiensis	89.12	34.00
23	NBRC 12165	GCA_001592205.1	B. vesicularis	95.24	61.50
24	NBRC 12165	GCA_001592205.1	B. vesicularis	95.16	61.50
25	DSM 17329	GCA_011761985.1	B. terrae	81.80	22.10
26	HAMBI_262	GCA_003350205.1	B. bullata	92.24	43.10
27	NBRC 12165	GCA_001592205.1	B. vesicularis	94.09	56.00
28	DSM 23858	GCA_000421705.1	B. naejangsanensis	92.13	44.00
29	ATCC 15264	GCA_000144605.1	B. subvibrioides	81.17	21.10

Virulence factors of Brevundimonas.

As strain 090558T was recovered from a human blood culture and Brevundimonas is known as an opportunistic pathogen (14), we attempted to identify potential virulence factors of 090558T 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-002T 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 (090558T). 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 20–22). 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 090558T 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 090558T. 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 β-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 090558T 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 090558T 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 090558T 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 090558T was performed on the Illumina HiSeq X10 platform (Illumina, San Diego, CA, USA). JCM 11415T, the type strain of B. nasdae, had >99% 16S rRNA sequence identity with strain 090558T but had no genome sequences or sequences of housekeeping genes for comparison. We therefore obtained B. nasdae strain JCM 11415T 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 090558T 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.

Whole-genome sequences of type strains of all Brevundimonas species (Table 2) were retrieved from GenBank. These genome sequences were annotated using Prokka v1.12 (31). Orthologues of these strains were identified using PIRATE v1.0.3 (https://github.com/SionBayliss/PIRATE) to represent the core genome. The gene sequences of the core genome were aligned and concatenated using MAFFT v7.313 (32) and AMAS v0.98 (33). A phylogenomic tree based on core genome sequences was then inferred using RAxML v8.2.12 (28) with the general-time-reversible (GTR) model plus gamma distribution and a 1,000-bootstrap test.

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 090558T were observed with an H-7650 transmission electron microscope (Hitachi, Tokyo, Japan). The growth of strain 090558T 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.

MICs of amikacin, ampicillin, ampicillin-sulbactam, aztreonam, ceftriaxone, ceftazidime, cefepime, cefotaxime, cefuroxime, chloramphenicol, ciprofloxacin, colistin, imipenem, meropenem, piperacillin-tazobactam, sulfamethoxazole-trimethoprim, and tigecycline were determined by Vitek II using the broth microdilution method. Breakpoints defined by the CLSI (34) were applied, except for tigecycline, for which breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/) were used.

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 090558T and B. nasdae JCM 11415T 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 090558T have been deposited in the DDBJ/EMBL/GenBank database under accession no. JABBJE000000000 and MT444005, respectively. The draft genome sequence of JCM 11415T has been deposited in the DDBJ/EMBL/GenBank database under accession no. JAEPWN000000000.