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. 2021 May 26;6(3):e00237-21. doi: 10.1128/mSystems.00237-21

Precise Species Identification for Acinetobacter: a Genome-Based Study with Description of Two Novel Acinetobacter Species

Jiayuan Qin a,b, Yu Feng a,b,c, Xiaoju Lü a,b, Zhiyong Zong a,b,c,d,
Editor: Rup Lale
PMCID: PMC8269215  PMID: 34061620

ABSTRACT

The genus Acinetobacter comprises species with ecological significance and opportunistic pathogens and has a complicated taxonomy. Precise species identification is a foundation for understanding bacteria. In this study, we found and characterized two novel Acinetobacter species, namely, Acinetobacter tianfuensis sp. nov. and Acinetobacter rongchengensis sp. nov., based on phenotype examinations and genome analyses of the two strains WCHAc060012T and WCHAc060115T. The two strains had ≤89.69% (mean, 79.28% or 79.72%) average nucleotide identity (ANI) and ≤36.4% (mean, 20.89% or 22.19%) in silico DNA-DNA hybridization (isDDH) values compared with each other and all known Acinetobacter species. Both species can be differentiated from all hitherto known Acinetobacter species by a combination of phenotypic characteristics. We found that Acinetobacter pullorum B301T and Acinetobacter portensis AC 877T are actually the same species with 98.59% ANI and 90.4% isDDH values. We then applied the updated taxonomy to curate 3,956 Acinetobacter genomes in GenBank and found that 6% of Acinetobacter genomes (n = 234) are required to be corrected or updated. We identified 56 novel tentative Acinetobacter species, extending the number of Acinetobacter species to 144, including 68 with species names and 76 unnamed taxa. We also found that ANI and the average amino acid identity (AAI) values among type or reference strains of all Acinetobacter species and taxa are ≥76.97% and ≥66.5%, respectively, which are higher than the proposed cutoffs to define the genus boundary. This study highlights the complex taxonomy of Acinetobacter as a single genus and the paramount importance of precise species identification. The newly identified unnamed taxa warrant further studies.

IMPORTANCE Acinetobacter species are widely distributed in nature and are of important ecological significance and clinical relevance. In this study, first, we significantly update the taxonomy of Acinetobacter by reporting two novel Acinetobacter species, namely, Acinetobacter tianfuensis and Acinetobacter rongchengensis, and by identifying Acinetobacter portensis as a synonym of Acinetobacter pullorum. Second, we curated Acinetobacter genome sequences deposited in GenBank (n = 3,956) using the updated taxonomy by correcting species assignations for 6% (n = 234) genomes and by assigning 94 (2.4%) to 56 previously unknown tentative species (taxa). Therefore, after curation, we further update the genus Acinetobacter to comprise 144 species, including 68 with species names and 76 unnamed taxa. Third, we addressed the question of whether such a large number of species should be divided in different genera and found that Acinetobacter is indeed a single genus. Our study significantly advanced the taxonomy of Acinetobacter, an important genus with science and health implications.

KEYWORDS: Acinetobacter, genome analysis, phylogenetic analysis, quasispecies, species

INTRODUCTION

The genus Acinetobacter, first proposed by Brisou and Prévot (1), is a highly diverse group. Members of the genus Acinetobacter are distributed widely in soil and water (2) and possess versatile metabolic capabilities for the degradation of various compounds, such as long-chain dicarboxylic acids and aromatics, and actively participate in the nutrient cycle in the ecosystem (3, 4). Some Acinetobacter species are also well-known opportunistic pathogens causing a variety of human infections (58). Precise species assignation lays a foundation for understanding the habitat, epidemiology, pathogenesis, and microbiological features of bacteria and has important implications for health, industry, and science, while updated and curated taxonomic assignment is the premise of precise species identification (9, 10). Before the present study, the genus Acinetobacter included 67 species with validly published names (11) and 20 additional Acinetobacter species with tentative species designations (www.szu.cz/anemec/Classification.pdf). Validly published names refer to those published in the International Journal of Systematic and Evolutionary Microbiology, the official journal of the International Committee on Systematics of Prokaryotes, including its validation lists (12). New Acinetobacter species are continuingly being reported, and the number of Acinetobacter species increases every year, with 6 novel species in 2017, 3 in 2018, 4 in 2019. and 9 in 2020 (11, 1318). However, the taxonomy of Acinetobacter is complicated by the presence of synonyms (1922). In addition, it is not uncommon that bacterial genomes deposited in GenBank are mislabeled for species assignations (10, 23, 24) (https://help.ezbiocloud.net/type-strain-and-reference-strain/). Therefore, there is a need to update the taxonomy of Acinetobacter and to curate the species assignations of Acinetobacter genome sequences deposited in GenBank.

Here, we report two novel Acinetobacter species, namely, Acinetobacter tianfuensis sp. nov. and Acinetobacter rongchengensis sp. nov., based on phenotypic characterization and genomic analysis. We updated the Acinetobacter taxonomy and found a pair of synonyms, Acinetobacter pullorum and Acinetobacter portensis, which has not been identified before. We then used the updated taxonomy to curate 5,997 Acinetobacter genomes available in GenBank (accessed by 1 August 2020), and we identified 56 previously unknown tentative species designations.

RESULTS

Identification of two novel Acinetobacter species, namely, Acinetobacter tianfuensis and Acinetobacter rongchengensis.

Two Acinetobacter strains, namely, WCHAc060012T and WCHAc060115T, were recovered from hospital sewage using an Acinetobacter chromogenic agar plate in 2018. We obtained the nearly complete 16S rRNA gene sequences (1,352 bp) of the two strains using PCR with the universal primers 27F and 1492R (25) and Sanger sequencing as described previously (26) for preliminary species identification. Comparison of the 16S rRNA gene sequences in the EzBioCloud database (27) and the 16S rRNA gene sequence-based phylogenetic tree (see Fig. S1 in the supplemental material) revealed that the two strains indeed belonged to the genus Acinetobacter. Strains WCHAc060012T and WCHAc060115T had the highest identity of 16S rRNA gene sequences with Acinetobacter chengduensis WCHAc060005T (98.96%; accession no. MK796535) and Acinetobacter chinensis WCHAc010005T (98.05%; accession no. NR_165666), respectively. However, it is well known that analysis based on the 16S rRNA sequence is insufficient for accurate taxonomic assignment (28). We then compared partial rpoB sequences (861 bp) of the two strains with those of Acinetobacter type strains. The two strains were also distinct from all known Acinetobacter species and formed two evolutionary clades in the phylogenetic tree based on partial rpoB gene sequences (see Fig. S2 in the supplemental material). Strain WCHAc060012T had the highest identity of the partial rpoB sequence with Acinetobacter wanghuae dk386T (89.08%), while WCHAc060115T had the highest identity with Acinetobacter piscicola KCTC 62134T (95.23%).

FIG S1

Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences (1,352 bp) of WCHAc060012T, WCHAc060115T, and type strains of Acinetobacter species with validly published names. The sequence of Moraxella lacunata ATCC 17967T (GenBank accession no. AF005160) was used as the outgroup. Bootstrap values (≥50%) after 1,000 resamplings are indicated at branch nodes. Shown in parentheses are the DDBJ/ENA/GenBank accession no. for 16S rRNA gene sequences or whole-genome sequences. Bar, 0.2 substitutions per nucleotide position. Download FIG S1, PDF file, 2.6 MB (2.7MB, pdf) .

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FIG S2

Maximum likelihood phylogenetic tree based on partial rpoB (861 bp) gene sequences of WCHAc060012T, WCHAc060115T, and type strains of Acinetobacter species with validly published names. Evolutionary distances were computed using Kimura’s two-parameter model. Moraxella lacunata ATCC 17967T was used as the outgroup. Bootstrap values ≥ 50% based on 1,000 resamplings are shown. Shown in parentheses are the DDBJ/ENA/GenBank accession no. for rpoB gene sequences or whole-genome sequences. Bar, 0.2 substitutions per nucleotide position. Download FIG S2, PDF file, 2.3 MB (2.3MB, pdf) .

Copyright © 2021 Qin et al.

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To further explore their precise species assignations, the two strains were subjected to whole-genome sequencing using Illumina HiSeq X10 platform. For strain WCHAc060012T, 6,401,206 reads and 1.92 giga-bases (Gb) were generated with an actual 549.9× coverage, which were assembled into a 3.5-Mb draft genome sequence containing 116 contigs (N50, 77,978 bp) with a G+C content of 42.3% . For strain WCHAc060115T, 5,479,547 reads and 1.64 Gb were generated with an actual 396.8 × coverage, which were assembled into a 4.1-Mb draft genome sequence containing 248 contigs (N50, 68,539 bp) with a G+C content of 37.7%. We determined the average nucleotide identity (ANI) values between WCHAc060012T and WCHAc060115T and between the two strains and type strains of all Acinetobacter species. Compared with type strains of all Acinetobacter species, ANI values of WCHAc060012T ranged from 77.09% (Acinetobacter puyangensis ANC 4466T) to 82.70% (Acinetobacter cumulans WCHAc060092T), while those of WCHAc060115T ranged from 77.71% (A. puyangensis ANC 4466T) to 89.69% (Acinetobacter piscicola LW15T) (Table 1). The ANI value between WCHAc060012T and WCHAc060115T was 79.46% (Table 1). These ANI values are well below the 95% to 96% threshold used to define bacterial species (29). We then performed in silico DNA-DNA hybridization (isDDH) analyses for WCHAc060012T, WCHAc060115T, and type strains of all Acinetobacter species. The isDDH values of WCHAc060012T and type strains of all Acinetobacter species were 19.2% to 23.4%, while those of WCHAc060115T and type strains of all Acinetobacter species were 20.0% to 36.4% (Table 1), which are far below the 70% cutoff used to define a species (30, 31). The isDDH value between WCHAc060012T and WCHAc060115T was 21.7% (Table 1). Both ANI and isDDH analyses clearly indicate that the two strains represent two novel Acinetobacter species. In the phylogenomic tree based on core genes (Fig. 1), WCHAc060012T and WCHAc060115T are most closely related to A. cumulans WCHAc060092T and A. piscicola LW15T, respectively.

TABLE 1.

Average nucleotide identity based on BLAST and in silico DNA-DNA hybridization values

Acinetobacter species and strain Accession no. ANI (%)/isDDH (%)a of:
GC content (%)
WCHAc060012T WCHAc060115T
A. albensis ANC 4874T FMBK00000000.1 79.15/20.2 79.48/20.9 38.4
A. apis ANC 5114T FZLN00000000.1 77.71/20.3 77.99/20.0 38.3
A. baumannii ATCC 19606T APRG00000000.1 78.75/19.9 79.37/21.1 39.1
A. baylyi CIP 107474T APPT00000000.1 78.47/19.7 78.91/20.7 40.4
A. beijerinckii CIP 110307T APQL00000000.1 78.46/20.8 79.42/21.1 38.3
A. bereziniae CIP 70.12T APQG00000000.1 79.26/21.1 82.98/27.1 38.2
A. bohemicus ANC 3994T APOH00000000.1 79.70/21.1 80.04/21.8 39.6
A. boissieri ANC 4422T FMYL00000000.1 77.81/19.5 77.79/20.1 38.0
A. bouvetii CIP 107468T APQD00000000.1 81.52/22.4 79.36/20.7 45.0
A. brisouii CIP 110357T AYEU00000000.1 78.70/21.7 79.13/22.3 41.7
A. calcoaceticus DSM 30006T APQI00000000.1 78.82/20.2 78.99/21.4 38.6
A. celticus ANC 4603T MBDL00000000.1 80.30/20.8 79.77/21.0 39.3
A. chengduensis WCHAc060005T RCHC00000000.1 81.99/22.2 79.52/21.8 39.9
A. chinensis WCHAc010005T CP032134.1 79.78/21.0 80.09/22.9 42.4
A. colistiniresistens NIPH 2036T ATGK00000000.1 78.72/20.5 81.37/28.6 41.0
A. courvalinii ANC 3623T APSA00000000.1 78.74/20.6 79.00/21.0 42.0
A. cumulans WCHAc060092T PYIW00000000.1 82.70/23.4 79.56/21.9 40.2
A. defluvii WCHA30T MAUF00000000.1 80.03/21.7 83.08/27.6 38.0
A. dispersus ANC 4105T APRL00000000.1 78.86/20.2 79.19/21.3 40.4
A. equi 114T CP012808.1 79.94/21.4 79.86/21.9 34.9
A. gandensis ANC 4275T LZDS00000000.1 81.09/21.4 79.94/21.4 39.7
A. gerneri CIP 107464T APPN00000000.1 79.48/22.5 80.13/22.9 37.7
A. guerrae AC 1271T LXGN00000000.1 78.65/19.2 78.89/20.1 39.2
A. guillouiae CIP 63.46T APOS00000000.1 79.10/21.3 82.02/24.6 38.2
A. gyllenbergii CIP 110306T ATGG00000000.1 78.52/20.1 79.35/22.5 40.8
A. haemolyticus CIP 64.3T APQQ00000000.1 78.95/21.5 79.22/22.3 39.7
A. halotolerans JCM 31009T SGIM00000000.1 78.58/19.8 79.02/20.5 40.0
A. harbinensis HITLi7T JXBK00000000.1 79.01/19.9 79.18/21.1 40.9
A. indicus CIP 110367T AYET00000000.1 79.99/21.3 79.69/21.5 45.4
A. johnsonii CIP 64.6T APON00000000.1 80.58/21.6 80.03/22.6 41.5
A. junii CIP 107470T APPS01000079.1 79.07/21.1 79.11/21.6 38.8
A. kookii ANC 4667T FMYO00000000.1 80.35/21.2 79.78/20.9 43.0
A. lactucae NRRL B-41902T LRPE00000000.1 78.85/19.8 78.98/21.3 38.6
A. lanii 185T CP049916.1 79.66/22.2 79.76/21.8 41.3
A. larvae BRTC-1T CP016895.1 78.06/20.6 78.21/21.8 41.6
A. lwoffii NIPH 512T AYHO00000000.1 80.01/21.3 79.12/22.0 43.0
A. modestus NIPH 236T APOJ00000000.1 78.72/20.3 79.53/21.9 38.4
A. nectaris CIP 110549T AYER00000000.1 77.98/20.1 78.08/20.5 36.7
A. nosocomialis NIPH 2119T APOP00000000.1 78.72/20.0 79.22/21.4 38.7
A. parvus CIP 108168T APOM00000000.1 79.12/21.8 79.18/22.3 41.7
A. piscicola KCTC 62134T NIFO00000000.1 79.33/21.0 89.69/36.4 37.2
A. pittii ATCC 19004T APQP01000014.1 78.73/20.2 78.98/21.2 38.8
A. populi PBJ7T NEXX00000000.1 77.54/20.6 78.02/21.1 40.2
A. portensis AC 877T LWRV00000000.1 80.10/21.2 80.14/21.8 36.6
A. pragensis ANC 4149T LUAW00000000.1 81.32/22.1 79.09/20.5 44.0
A. proteolyticus NIPH 809T APOI00000000.1 78.57/19.9 79.80/22.2 41.1
A. pseudolwoffii ANC 5044T PHRG00000000.1 80.14/21.0 79.42/21.3 43.3
A. pullicarnis S23T VCMZ00000000.1 78.10/21.7 79.92/24.9 41.5
A. pullorum B301T JAAARQ000000000.1 80.21/21.5 80.14/22.3 37.0
A. puyangensis ANC 4466T OANT00000000.1 77.09/20.1 77.71/20.5 40.2
A. qingfengensis ANC 4671T MKKK00000000.1 77.52/19.9 77.88/21.0 38.1
A. radioresistens DSM 6976T APQF00000000.1 78.59/19.8 78.78/20.8 41.7
A. rongchengensis WCHAc060115T RAXT00000000.1 79.46/21.7 37.7
A. rudis CIP 110305T ATGI00000000.1 78.27/20.8 78.90/21.0 39.0
A. schindleri CIP 107287T APPQ00000000.1 80.38/21.4 79.65/21.7 42.2
A. seifertii NIPH 973T APOO00000000.1 78.94/20.7 79.35/22.6 38.6
A. shaoyimingii 323-1T CP049801.1 79.61/22.3 79.92/21.7 38.3
A. sichuanensis WCHAc060041T PYIX00000000.1 79.86/21.9 83.12/27.2 37.2
A. soli CIP 110264T APPU00000000.1 78.28/19.7 78.64/20.2 43.2
A. tandoii CIP 107469T AQFM00000000.1 79.89/20.5 80.58/23.2 40.0
A. tianfuensis WCHAc060012T RAXV00000000.1 79.46/21.7 42.3
A. tjernbergiae CIP 107465T AYEV00000000.1 78.79/20.1 79.63/22.0 38.5
A. towneri CIP 107472T APPY00000000.1 80.03/21.7 79.97/21.9 41.2
A. ursingii CIP 107286T APQA00000000.1 78.73/19.9 79.25/22.1 40.1
A. variabilis NIPH 2171T APRS00000000.1 79.95/20.9 79.76/22.4 42.0
A. venetianus CIP 110063T APPO00000000.1 78.77/20.4 79.11/20.8 39.1
A. vivianii NIPH 2168T APRW00000000.1 78.90/20.6 79.20/21.3 41.4
A. wanghuae dk386T CP045650.1 79.88/21.0 79.65/21.0 40.6
A. wuhouensis WCHA60T MBPR00000000.1 79.95/22.1 82.04/24.0 38.1
a

ANI and isDDH values were calculated using fastANI v1.32 (46) and the genome-to-genome distance calculator (formula 2) (47), respectively.

FIG 1.

FIG 1

Phylogenomic tree of WCHAc060012T, WCHAc060115T, and type strains of Acinetobacter species with validly published names. The phylogenomic tree was inferred based on the alignment of 1,397 core genes. WCHAc060012T and WCHAc060115T are highlighted in bold. A. pullorum and A. portensis, a pair of synonyms, are highlighted in red. DDBJ/ENA/GenBank accession no. are shown in parentheses and 100% bootstraps are indicated. Bar, 0.2 changes per nucleotide position.

After phenotypic characterizations (see below), we propose strain WCHAc060012T with the name Acinetobacter tianfuensis sp. nov. (tian.fu.en’sis. N.L. masc. adj. tianfuensis, referring to Chengdu City, Sichuan Province, China) and WCHAc060115T with the name Acinetobacter rongchengensis sp. nov. (rong.cheng.en’sis. N.L. masc. adj. rongchengensis, another name referring to Chengdu City, Sichuan Province, China). The type strain of Acinetobacter tianfuensis and Acinetobacter rongchengensis is WCHAc060012T (=GDMCC 1.1623T =JCM 33510T) and WCHAc060115T (=GDMCC 1.1625T =JCM 33512T), respectively.

The two novel Acinetobacter species may be able to be differentiated from other Acinetobacter species by a combination of phenotypic characteristics.

The phenotypic characteristics tested using the genus-targeted set of physiological and metabolic tests are presented in the standard way used in previous nomenclatural proposals (32, 33). The phenotypes for the two novel Acinetobacter species, together with those for all known Acinetobacter species with validly published names, are summarized in Data Set S1 in the supplemental material. For both strains, growth occurs at various pHs from 7 to 8 and the temperatures range 20 to 35°C. Strain WCHAc060012T grows at 30°C in the presence of 0% to 3% (wt/vol) NaCl in tryptic soy broth (TSB), while WCHAc060115T grows in 0% to 4% (wt/vol) NaCl. Both strains were positive for the catalase test but negative for the oxidase activity. Cells of the two strains are Gram-negative coccobacilli; strictly aerobic; nonsporogenous; incapable of swimming motility; and capable of growing on media such as tryptic soy agar (TSA), Luria-Bertani (LB) agar, BHI agar, and Müller-Hinton agar (all from Hopebio). Colonies are light yellow, circular, opaque, smooth, convex, with entire margins, and approximately 1.0 to 2.0 mm in diameter after 24 h of incubation at 30°C on BHI agar plates.

DATA SET S1

Phenotypic properties of A. tianfuensis sp. nov., A. rongchengensis sp. nov. and the Acinetobacter species with validly published names. Download Data Set S1, XLSX file, 0.03 MB (30.3KB, xlsx) .

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Phenotypic differences between the two novel Acinetobacter species and each of the known species with validly published names are indicated in Data Set S1. When considering only clearly positive or clearly negative results, the most useful combinations of characteristics for differentiating WCHAc060012T from all known Acinetobacter species include growth on l-glutamate, d-malate, malonate, and phenylacetate but no growth on l-arabinose, l-arginine, azelate, and glutarate (Data Set S1). Strain WCHAc060115T could be differentiated from all known Acinetobacter species by the combination of assimilation trans-aconitate, citrate (Simmons’), and l-tartrate but not β-alanine and 4-aminobutyrate (Data Set S1).

We also identified antimicrobial resistance genes from genome sequences of the two strains (see Table S1 in the supplemental material). Both strains had genes mediating resistance to aminoglycosides, sulfonamides, and macrolides, while WCHAc060115T also harbored two carbapenemase genes, namely, blaNDM-1 and blaOXA-58, and WCHAc060012T carried a tetracycline-resistant gene tet(39).

TABLE S1

Antimicrobial resistance genes of A. tianfuensis WCHAc060012T and A. rongchengensis WCHAc060115T. Download Table S1, PDF file, 0.1 MB (71.7KB, pdf) .

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Acinetobacter pullorum Elnar et al. 2020 and Acinetobacter portensis Ana et al. 2020 are the same species.

During the process of studying WCHAc060012T and WCHAc060115T, we also found a pair of synonyms, namely, A. pullorum and A. portensis. A. pullorum B301T was isolated from raw chicken meat at a local market in Korea (14). It has been shown that A. pullorum B301T is closely related to Acinetobacter celticus ANC 4603T (14). Four A. portensis strains were isolated from raw meat samples in supermarkets in Porto, Portugal. A. portensis is also closely related to A. celticus ANC 4603T (15). A comparison of the 16S rRNA gene sequences for the two type strains showed a 99.70% similarity. The draft genome sequence of A. pullorum B301T (GenBank accession no. JAAARQ000000000) and that of A. portensis AC 877T (GenBank accession no. LWRV00000000) have a 90.4% isDDH value and a 98.59% ANI value. Both ANI and isDDH analyses clearly indicate that the two species are actually the same species. In the phylogenomic tree, A. pullorum B301T and A. portensis AC 877T indeed cluster together (Fig. 1, highlighted in red).

A comparison of the physiological and biochemical features of the two type strains shows phenotype coherence, which is summarized in Table S2 in the supplemental material. According to previous reports (14), A. pullorum and A. portensis are different in the acidification of d-glucose and utilization of β-alanine and d-glucose, which is likely due to intraspecies variability or assay conditions. Based on principles by the International Code of Nomenclature of Bacteria (12), A. pullorum has the priority of species name over A. portensis. We therefore propose that A. portensis (15) is a later heterotypic synonym of A. pullorum (14).

TABLE S2

The characteristics of strains of A. pullorum and A. portensis. Download Table S2, PDF file, 0.1 MB (143.5KB, pdf) .

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Curation of Acinetobacter genomes with the updated taxonomy.

Based on the above findings, the valid species names of Acinetobacter should be updated to comprise 68 species at present (Table 2). In addition, there are 20 tentative species designations of Acinetobacter (www.szu.cz/anemec/Classification.pdf) (Table 2). We then applied the updated Acinetobacter taxonomy to curate the 5,997 Acinetobacter strains with genome sequences deposited in GenBank (accessed by 1 August 2020). Before curation, we performed a quality-control check for all of the genomes. Among the 5,997 genomes, 2,041 were discarded due to low quality defined by >300 contigs for individual genomes (n = 444), a <50-kb N50 value (n = 458), <90% genome completeness (n = 20), genome contamination (n = 206), or genome heterogeneity (n = 913). We then used the remaining 3,956 genomes for precise species identification by both ANI and isDDH. Among the 3,956 Acinetobacter genomes, 3,777 were labeled with a known Acinetobacter species name (Data Set S2). The remaining 179 strains were labeled only with Acinetobacter sp. (n = 175), Acinetobacter genomosp. (n = 2), Acinetobacter calcoaceticus/Acinetobacter baumannii complex (n = 1), or uncultured Acinetobacter (n = 1) (Data Set S2), which were updated by our curation. Species were misidentified for 55 Acinetobacter genomes (Data Set S2 and summarized in Table S3 in the supplemental material). The 55 misidentified genomes include 13 labeled with A. baumannii but actually belonging to other Acinetobacter species and four of non-A. baumannii Acinetobacter species actually belonging to other closely related species (Table S3), while the remaining 38 genomes should be assigned to novel taxa (see below for details). Therefore, there were 234 genomes whose species identification needs to be corrected (n = 55) or updated (n = 179) according to the findings in this study (Data Set S2).

TABLE 2.

Updated classification and nomenclature of the genus Acinetobacter before species curation for genomes in GenBank

Species name Type strain or reference strain Accession no.
Valid (n = 68)
    Acinetobacter albensis ANC 4874T FMBK00000000
    Acinetobacter apis ANC 5114T FZLN00000000
    Acinetobacter baumannii CIP 70.34T APRG00000000
    Acinetobacter baylyi CIP 107474T APPT00000000
    Acinetobacter beijerinckii CIP 110307T APQL00000000
    Acinetobacter bereziniae CIP 70.12T APQG00000000
    Acinetobacter bohemicusa ANC 3994T APOH00000000
    Acinetobacter boissieri ANC 4422T FMYL00000000
    Acinetobacter bouvetii CIP 107468T APQD00000000
    Acinetobacter brisouii ANC 4119T APPR00000000
    Acinetobacter calcoaceticus CIP 81.8T APQI00000000
    Acinetobacter celticus ANC 4603T MBDL00000000
    Acinetobacter chengduensis WCHAc060005T RCHC00000000
    Acinetobacter chinensis WCHAc010005T CP032134
    Acinetobacter colistiniresistens NIPH 2036T ATGK00000000
    Acinetobacter courvalinii ANC 3623T APSA00000000
    Acinetobacter cumulans WCHAc060092T PYIW00000000
    Acinetobacter defluvii WCHA30T CP029397
    Acinetobacter dispersus ANC 4105T APRL00000000
    Acinetobacter equi 114T CP012808
    Acinetobacter gandensis ANC 4275T LZDS00000000
    Acinetobacter gerneri CIP 107464T APPN00000000
    Acinetobacter guerrae AC 1271T LXGN00000000
    Acinetobacter guillouiae CIP 63.46T APOS00000000
    Acinetobacter gyllenbergii CIP 110306T ATGG00000000
    Acinetobacter haemolyticus CIP 64.3T APQQ00000000
    Acinetobacter halotolerans JCM 31009T SGIM00000000
    Acinetobacter harbinensis HITLi7T JXBK00000000
    Acinetobacter indicusb ANC 4215T ATGH00000000
    Acinetobacter johnsonii CIP 64.6T APON00000000
    Acinetobacter juniic CIP 64.5T APPX00000000
    Acinetobacter kookii ANC 4667T FMYO00000000
    Acinetobacter lactucaed NRRL B-41902T LRPE00000000
    Acinetobacter lanii 185T CP049916
    Acinetobacter larvae BRTC-1T CP016895
    Acinetobacter lwoffiie NIPH 512T AYHO00000000
    Acinetobacter modestus NIPH 236T APOJ00000000
    Acinetobacter nectaris CIP 110549T AYER00000000
    Acinetobacter nosocomialis NIPH 2119T APOP00000000
    Acinetobacter parvus CIP 108168T APOM00000000
    Acinetobacter piscicola LW15T NIFO00000000
    Acinetobacter pittii CIP 70.29T APQP00000000
    Acinetobacter populi PBJ7T NEXX00000000
    Acinetobacter pragensis ANC 4149T LUAW00000000
    Acinetobacter proteolyticus NIPH 809T APOI00000000
    Acinetobacter pseudolwoffii ANC 5044T PHRG00000000
    Acinetobacter pullicarnis S23T CP036789
    Acinetobacter pullorumf B301T JAAARQ000000000
    Acinetobacter puyangensis ANC 4466T OANT00000000
    Acinetobacter qingfengensis ANC 4671T MKKK00000000
    Acinetobacter radioresistens CIP 103788T APQF00000000
    Acinetobacter rongchengensis WCHAc060115T RAXT00000000
    Acinetobacter rudis CIP 110305T ATGI00000000
    Acinetobacter schindleri CIP 107287T APPQ00000000
    Acinetobacter seifertii NIPH 973T APOO00000000
    Acinetobacter shaoyimingii 323-1T CP049801
    Acinetobacter sichuanensis WCHAc060041T PYIX00000000
    Acinetobacter soli CIP 110264T APPU00000000
    Acinetobacter tandoii CIP 107469T AQFM00000000
    Acinetobacter tianfuensis WCHAc060012T RAXV00000000
    Acinetobacter tjernbergiae CIP 107465T AYEV00000000
    Acinetobacter towneri CIP 107472T APPY00000000
    Acinetobacter ursingii CIP 107286T APQA00000000
    Acinetobacter variabilis NIPH 2171T APRS00000000
    Acinetobacter venetianus CIP 110063T APPO00000000
    Acinetobacter vivianii NIPH 2168T APRW00000000
    Acinetobacter wanghuae dk386T CP045650
    Acinetobacter wuhouensis WCHA60T CP031716
Tentative designations (n = 20)
    Acinetobacter kyonggiensis ANC 5109 FNPK00000000
    Acinetobacter marinus ANC 3699 FMYK00000000
    Acinetobacter oleivorans DR1 CP002080
    Genomic sp. 6 CIP a165 APOK00000000
    Genomic sp. 15BJ CIP 110321 AQFL00000000
    Genomic sp. 16 CIP 70.18 APRN00000000
    Taxon 21 ANC 3929 APRH00000000
    Taxon 22 NIPH 2100 APSB00000000
    Taxon 24A ANC 4655 NEGF00000000
    Taxon 24B ANC 4471 SJNZ00000000
    Taxon 25A ANC 3789 APOY00000000
    Taxon 25B ANC 4633 SJNX00000000
    Taxon 27 ANC 4169 NEGE00000000
    Taxon 32 ANC 4218 NEGD00000000
    Taxon 34 ANC 4470 NEGC00000000
    Taxon 35 ANC 4999 NEGB00000000
    Taxon 36 ANC 4945 MVKX00000000
    Taxon 37 WCHAc010034 CP032279
    Taxon 38 ANC 3903 NEGA00000000
    Taxon 39 ANC 4204 NEFZ00000000
a

Acinetobacter pakistanensis is a later synonym of Acinetobacter bohemicus (57).

b

Acinetobacter guangdongensis is a later synonym of Acinetobacter indicus (20).

c

Acinetobacter grimontii is a later synonym of Acinetobacter junii (21).

d

Acinetobacter dijkshoorniae is a later synonym of Acinetobacter lactucae (22).

e

Acinetobacter mesopotamicus is a later synonym of Acinetobacter lwoffii (19).

f

Acinetobacter portensis is a later synonym of Acinetobacter pullorum (this study).

TABLE S3

The 55 Acinetobacter genome sequences with misidentified species in NCBI. Download Table S3, PDF file, 0.1 MB (92.7KB, pdf) .

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DATA SET S2

The 3,956 Acinetobacter genome sequences available in GenBank (accessed by 1 August 2020). Download Data Set S2, XLSX file, 0.4 MB (427.5KB, xlsx) .

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After precise species identification, among the 3,956 Acinetobacter strains with genome sequences available, most (n = 3,124, 79.0%) belonged to A. baumannii, followed by Acinetobacter pittii (n = 174, 4.4%), Acinetobacter nosocomialis (n = 103, 2.6%), and Acinetobacter indicus (n = 68, 1.7%; Table 3). However, 94 (2.4%) strains could not be assigned to any known Acinetobacter species nor to any known tentative species designations (Data Set S3). Instead, the 94 strains could be assigned to 56 potentially novel Acinetobacter species, which are named taxon 40 to 95 here (Table 4 and Fig. 2), as Acinetobacter taxon 39 has been used before. Characterization of taxon 40 to 95 by phenotype methods is warranted to further establish their species status with proper species names under current International Code of Nomenclature of Prokaryotes (12).

TABLE 3.

Species distribution of 3,956 Acinetobacter strains with genome sequences available in GenBank

Species No. of genomes Taxon without a species namea No. of genomes
Acinetobacter albensis 2 Genomic sp. 6 2
Acinetobacter apis 1 Genomic sp. 15BJ 1
Acinetobacter baumannii 3,124 Genomic sp. 16 6
Acinetobacter baylyi 11 Taxon 21 1
Acinetobacter beijerinckii 3 Taxon 22 1
Acinetobacter bereziniae 1 Taxon 24A 1
Acinetobacter bohemicus 1 Taxon 24B 3
Acinetobacter boissieri 1 Taxon 25A 2
Acinetobacter bouvetii 3 Taxon 25B 2
Acinetobacter brisouii 4 Taxon 27 1
Acinetobacter calcoaceticus 15 Taxon 32 1
Acinetobacter celticus 1 Taxon 34 1
Acinetobacter chengduensis 2 Taxon 35 1
Acinetobacter chinensis 2 Taxon 36 1
Acinetobacter colistiniresistens 4 Taxon 37 1
Acinetobacter courvalinii 5 Taxon 38 1
Acinetobacter cumulans 8 Taxon 39 2
Acinetobacter defluvii 2 Taxon 40 1
Acinetobacter dispersus 1 Taxon 41 1
Acinetobacter equi 1 Taxon 42 3
Acinetobacter gandensis 1 Taxon 43 2
Acinetobacter gerneri 2 Taxon 44 2
Acinetobacter guerrae 3 Taxon 45 5
Acinetobacter guillouiae 1 Taxon 46 4
Acinetobacter gyllenbergii 4 Taxon 47 2
Acinetobacter haemolyticus 20 Taxon 48 1
Acinetobacter halotolerans 1 Taxon 49 1
Acinetobacter harbinensis 1 Taxon 50 1
Acinetobacter indicus 68 Taxon 51 1
Acinetobacter johnsonii 8 Taxon 52 7
Acinetobacter junii 27 Taxon 53 2
Acinetobacter kookii 2 Taxon 54 5
Acinetobacter kyonggiensis 1 Taxon 55 1
Acinetobacter lactucae 7 Taxon 56 2
Acinetobacter lanii 2 Taxon 57 1
Acinetobacter larvae 1 Taxon 58 1
Acinetobacter lwoffii 17 Taxon 59 2
Acinetobacter marinus 1 Taxon 60 1
Acinetobacter modestus 2 Taxon 61 1
Acinetobacter nectaris 1 Taxon 62 3
Acinetobacter nosocomialis 103 Taxon 63 1
Acinetobacter oleivorans 7 Taxon 64 1
Acinetobacter parvus 8 Taxon 65 3
Acinetobacter piscicola 1 Taxon 66 4
Acinetobacter pittii 174 Taxon 67 1
Acinetobacter populi 1 Taxon 68 1
Acinetobacter pragensis 1 Taxon 69 1
Acinetobacter proteolyticus 5 Taxon 70 1
Acinetobacter pseudolwoffii 3 Taxon 71 3
Acinetobacter pullicarnis 1 Taxon 72 1
Acinetobacter pullorum 2 Taxon 73 1
Acinetobacter puyangensis 1 Taxon 74 1
Acinetobacter qingfengensis 2 Taxon 75 1
Acinetobacter radioresistens 32 Taxon 76 2
Acinetobacter rongchengensis 1 Taxon 77 1
Acinetobacter rudis 1 Taxon 78 1
Acinetobacter schindleri 10 Taxon 79 1
Acinetobacter seifertii 19 Taxon 80 1
Acinetobacter shaoyimingii 2 Taxon 81 1
Acinetobacter sichuanensis 1 Taxon 82 1
Acinetobacter soli 22 Taxon 83 3
Acinetobacter tandoii 2 Taxon 84 1
Acinetobacter tianfuensis 1 Taxon 85 1
Acinetobacter tjernbergiae 3 Taxon 86 2
Acinetobacter towneri 11 Taxon 87 1
Acinetobacter ursingii 29 Taxon 88 1
Acinetobacter variabilis 6 Taxon 89 1
Acinetobacter venetianus 10 Taxon 90 1
Acinetobacter vivianii 5 Taxon 91 1
Acinetobacter wanghuae 2 Taxon 92 1
Acinetobacter wuhouensis 6 Taxon 93 1
Taxon 94 1
Taxon 95 1
a

Taxa identified in this study are highlighted in bold.

TABLE 4.

Tentative taxon assignations for novel, unnamed Acinetobacter species identified in this study

Taxon Accession no. Reference straina Closest species or taxon ANI (%) isDDH (%)
40 GCA_000214135.2 P8-3-8 A. piscicola 88.77 34.4
41 GCA_000313935.1 WC-141 A. oleivorans 93.08 49.3
42 GCA_000368805.1 ANC 3681 A. johnsonii 95.83 66.6
43 GCA_000369485.1 ANC 4105 A. dispersus 95.61 62.4
44 GCA_000369765.1 NIPH 1859 A. colistiniresistens 95.29 60.3
45 GCA_000369425.1 NIPH 284 A. modestus 94.51 54.8
46 GCA_000368405.1 NIPH 817 A. oleivorans 95.2 61.3
47 GCA_000386005.1 MDS7A A. towneri 93.8 52.9
48 GCA_000399685.1 ANC 4050 A. lactucae 94.01 53.5
49 GCA_000399665.1 ANC 3811 A. oleivorans 94.28 55.8
50 GCA_000805455.1 A47 A. courvalinii 88 31.9
51 GCA_001432505.1 ABBL016 A. pittii 94.84 58
52 GCA_001483265.1 MB44 A. johnsonii 95.84 65.6
53 GCA_001510805.1 GK2 A. calcoaceticus 93.65 51.5
54 GCA_001592855.1 BMW17 A. johnsonii 95.6 64.9
55 GCA_001612555.1 TGL-Y2 A. bohemicus 80.2 22.1
56 GCA_001647535.1 SFA A. lwoffii 90.48 38.1
57 GCA_001647545.1 SFB Taxon 24B 89.6 36.8
58 GCA_900109815.1 DSM 11652 A. cumulans 80.48 21.5
59 GCA_002018395.1 ANC 5600 Taxon 36 95.48 62.6
60 GCA_002135375.1 ANC 4558 A. equi 81.73 23
61 GCA_002135345.1 ANC 4648 Taxon 35 87.82 33
62 GCA_002137095.1 PR366 A. pittii 95.08 59.4
63 GCA_002296655.1 UBA801 A. towneri 93.42 50.5
64 GCA_002365595.1 UBA3106 A. kookii 88.29 32.6
65 GCA_002795165.1 SC36 A. tandoii 87.13 29.9
66 GCA_003053325.1 KCJK7889 A. pittii 95.5 62.5
67 GCA_003105055.1 AM A. tandoii 92 43.2
68 GCA_900406815.1 KCRI-348C A. haemolyticus 92.5 46.7
69 GCA_003268395.1 CFCC 10889 A. wuhouensis 85.29 29.1
70 GCA_003687745.1 2JN-4 A. halotolerans 95.44 59.7
71 GCA_003711395.1 B51(2017) A. gandensis 80.94 21.9
72 GCA_003359215.2 2012N08-034 A. pittii 95.95 65.4
73 GCA_900625095.1 Marseille-P8049 A. ursingii 84.88 26.8
74 GCA_003939325.1 AJ_082 A. johnsonii 95.75 65.9
75 GCA_003952785.1 IC001 A. johnsonii 95.86 66.6
76 GCA_004152775.1 C1T1-2_a A. sichuanensis 86.05 28.5
77 GCA_004331035.1 ANC 4910 A. tandoii 91 40
78 GCA_004331175.1 ANC 4178 A. tandoii 91.25 40.6
79 GCA_004331185.1 ANC 4249 Taxon 24B 95.48 61
80 GCA_004336635.1 ANC 4862 Taxon 24A 92.69 47.2
81 GCA_004345325.1 JUb89 A. pullicarnis 79.81 21.2
82 GCA_004364945.1 3664 A. calcoaceticus 95.97 65.8
83 GCA_007570885.1 RF15A A. variabilis 83.02 24.6
84 GCA_008630915.1 C16S1 A. haemolyticus 93.7 50.1
85 GCA_009707625.1 YIM 103518 A. pullorum 87.32 30.6
86 GCA_009822135.1 SCsl29 A. variabilis 95.33 62.9
87 GCA_902809855.1 Marseille-Q1618 A. defluvii 91.33 42.3
88 GCA_902825285.1 Marseille-Q1620 A. gerneri 81.13 22.2
89 GCA_011753255.1 Tr-809 A. dispersus 91.34 41.3
90 GCA_902753875.1 SFB21 Taxon 32 85.48 27.8
91 GCA_012371315.1 A1 A. towneri 88.81 32.8
92 GCA_013004315.1 ANC 5378 Taxon 24A 92.8 47.2
93 GCA_013004295.1 ANC 5414 Taxon 24A 92.74 47
94 GCA_013004275.1 ANC 4277 Taxon 24A 95.98 64.1
95 GCA_013072695.1 Ac_5812 Genomic sp. 16 92.7 47.2
a

The strain with genome sequence deposited in GenBank at the earliest date was selected as the reference strain for the newly identified taxa.

FIG 2.

FIG 2

Phylogenomic tree of Acinetobacter species with validly published names and tentative taxa. The phylogenomic tree was inferred based on the alignment of 1,397 core genes. Strains and their nucleotide accession no. are listed alongside the names of species, and 100% bootstrap are shown. Bar, value indicates the nucleotide substitutions per site. The two novel Acinetobacter species are depicted in green, while novel Acinetobacter taxa identified in this study, namely, taxon 40 to 95, are in red.

DATA SET S3

The potential tentative species designation. Download Data Set S3, XLSX file, 0.02 MB (16.1KB, xlsx) .

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Acinetobacter is indeed a single genus comprising 144 species at present.

The identification of the 56 taxa also extends the number of Acinetobacter species to 144, including 68 with species names and 76 unnamed taxa. The large number of species raises the question whether Acinetobacter is indeed a single genus or actually should be divided into different genera. ANI values among type strains of all species and reference strains of all taxa of the genus Acinetobacter are ≥76.97% (76.97% to 95.98%) (see Data Set S4 in the supplemental material). The ANI values are higher than 72.50% to 73.70%, which has been proposed as the 95% confidence interval of the boundary to define a bacterial genus (34). To further verify the genus Acinetobacter, the average amino acid identity (AAI) values among type strains of all species and reference strains of all taxa of the genus Acinetobacter were also calculated, which are >66% (66.5% to 97.4%) (see Data Set S5 in the supplemental material). This is higher than the proposed cutoff of 65% AAI used to define a bacterial genus (34, 35). Both ANI and AAI analyses suggest that all Acinetobacter species and unnamed taxa identified so far indeed belong to a single genus.

DATA SET S4

Average nucleotide identity between type strains of Acinetobacter species and reference strains of Acinetobacter taxa without species names. Download Data Set S4, XLSX file, 0.1 MB (124.3KB, xlsx) .

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DATA SET S5

Average amino acid identity between type strains of Acinetobacter species and reference strains of Acinetobacter taxa without species names. Download Data Set S5, XLSX file, 0.1 MB (129.7KB, xlsx) .

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DISCUSSION

In this study, we first found and characterized two novel Acinetobacter species. We also found that A. pullorum and A. portensis are synonyms and then updated the taxonomy of the genus Acinetobacter. We applied the updated taxonomic assignments to curate genome sequences deposited in GenBank with the label of Acinetobacter and found that 6% (n = 234) of the 3,956 genomes with good quality need to be corrected or updated for species identification. We also identified 56 previously unknown tentative species designations, which further update the genus Acinetobacter to comprise 144 species, including 68 with species names and 76 taxa without species names. Such a large number of species raises the question whether Acinetobacter should be divided into multiple genera. Although the boundary of bacterial genera based on genome sequences is less established than that of species and requires more studies (9), our ANI and AAI analyses suggest that all Acinetobacter species indeed belong to a single genus. The mechanisms and factors driving the divergence of Acinetobacter to form the evolutionary trajectory and generate the remarkable species diversity and form have not been understood (36, 37).

Along with many Acinetobacter species identified recently (11, 1318), the above findings highlight that Acinetobacter is a highly diverse and complex group (38). The species status of two novel Acinetobacter species, namely, A. tianfuensis and A. rongchengensis, was established by both genome- and phenotype-based methods. In addition to known species, there were 76 tentative novel Acinetobacter taxa, including 56 identified in this study. The identification of new taxa invites more studies on these tentative species by both genome- and phenotype-based methods to establish their species status and to propose appropriate species names under the current code for prokaryotes (12). Alternatively, it has also been proposed to create a new code that would use DNA sequences as the type material to rule the nomenclature of prokaryotes (39) or to establish placeholder species names using genome-based taxonomy (10). Indeed, there is an urgent need to find a solution to deal with the exploration of new taxonomic findings generated by genome sequencing (40).

In conclusion, we characterized and reported two novel Acinetobacter species, namely, A. tianfuensis and A. rongchengensis. A. tianfuensis may be distinguished from all other Acinetobacter species by its ability to grow on l-glutamate, d-malate, malonate, and phenylacetate but not grow on l-arabinose, l-arginine, azelate, and glutarate. A. rongchengensis may be differentiated from all other Acinetobacter species by the combination of assimilation trans-aconitate, citrate (Simmons’), and l-tartrate but not β-alanine and 4-aminobutyrate. We also found that A. portensis is a later heterotypic synonym of A. pullorum. We demonstrated that some Acinetobacter genome sequences deposited in GenBank are required to be corrected and identified 56 novel tentative Acinetobacter taxa, which warrant further phenotype-based characterizations.

MATERIALS AND METHODS

Strains and preliminary species identification.

Hospital sewage (1 ml) was collected from the influx mainstream of the wastewater treatment plant at West China Hospital in June 2018, which was added in 10 ml nutrient broth (Oxoid, Basingstoke, UK) and was incubated overnight at 30°C with shaking. The culture suspension was diluted to 0.5 McFarland standard and was then further diluted to 1:100 with saline. A 100-μl aliquot was then streaked onto an Acinetobacter chromogenic agar plate (CHROMagar, Paris, France). The plate was then incubated at 30°C overnight. All isolates recovered from the plate were subjected to preliminary species identification by partial sequencing of the RNA polymerase β subunit-encoding rpoB gene using PCR and Sanger sequencing as described previously (5). Isolates with ≤98% identity of the 861-bp partial rpoB sequence (corresponding to nucleotide positions 2915 to 3775 of A. baumannii CIP 70.34T; accession no. DQ207471) to type strains of all known Acinetobacter species may belong to novel species and were characterized as described below. Two Acinetobacter isolates, namely, WCHAc060012T and WCHAc060115T, were recovered from the plate and had ≤98% identity of the 861-bp partial rpoB sequence to type strains of all known Acinetobacter species.

Analysis based on 16S rRNA and rpoB genes.

Boiled lysates were used as the PCR template, and PCR amplicons were sequenced using the Sanger method (26). The nearly complete 16S rRNA gene sequences of WCHAc060012T and WCHAc060115T were obtained using PCR with universal primers 27F and 1492R (25). The 16S rRNA gene sequences of type strains of each Acinetobacter species were retrieved from their depositions in GenBank or from their whole-genome sequences. The longest common fragments of the 16S rRNA gene sequences (1,352 bp) were aligned using MAFFT v7.471 (41), and a maximum likelihood phylogenetic tree (42) based on the 1,352-bp sequences was inferred using RAxML v8.2.12 (43) with the general time reversible (GTR) model.

To further investigate the taxonomic position of WCHAc060012T and WCHAc060115T, 861-bp partial rpoB sequences of type strains of each Acinetobacter species were retrieved from their depositions in GenBank or from their whole-genome sequences. Sequence alignment and the construction of a maximum-likelihood phylogenetic tree were performed as described above.

Whole-genome sequencing of the two strains.

Genomic DNA from an overnight culture of each of the two strains was prepared using the QIAamp DNA minikit (Qiagen, Hilden, Germany) and was then subjected to whole-genome sequencing using the HiSeq X10 sequencing platform (Illumina, San Diego, CA, USA) with an approximate 250× coverage. Reads were de novo assembled into contigs using the program SPAdes v3.15.1 (44). Potential contaminations of WCHAc060012T and WCHAc060115T genomes were checked using CheckM v1.1.3 (45). Antimicrobial resistance genes were identified from genome sequences using the ABRicate program (https://github.com/tseemann/abricate) to query the ResFinder database 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/).

Precise species identification and phylogenomic analysis of the two strains.

Whole-genome sequences of type strains of all Acinetobacter species (Data Set S2) were retrieved from the NCBI database. Genome sequences of WCHAc060012T and WCHAc060115T were compared with those of type strains of Acinetobacter species using the average nucleotide identity based on BLAST (ANI) and in silico DNA-DNA hybridization (isDDH). ANI and isDDH values were calculated using the fastANI v1.32 (46) and genome-to-genome distance calculator (formula 2) (47) with the recommended parameters and/or default settings, respectively. A ≥96% ANI (31) or ≥70.0% isDDH (31, 47) was used as the cutoff to define a bacterial species.

A core genome phylogenetic tree based on concatenated sequences of core genes was constructed as described previously (48). Prokka v1.14.5 (49) and Prodigal v2.6.3 (50) were used to annotate these genome sequences, and protein-encoding sequences for each genome were retrieved for gene alignment and clustering using PIRATE v1.0.4 (51). The gene sequences were aligned and concatenated using MAFFT v7.471 (41) and AMAS v0.98 (52), which were then used to infer a phylogenomic tree using RAxML v8.2.12 (43) with GTR model plus gamma distribution and a 1,000-bootstrap test. The phylogenetic tree was visualized with FigTree (https://github.com/rambaut/figtree).

Phenotypic characterization for strains of two novel species.

The metabolic and physiological properties were assessed using the standardized genus-targeted set of metabolic/physiological tests as described previously (5, 32, 53). The two strains were grown on brain heart infusion (BHI) agar (Oxoid) plates at 30°C overnight, and the colony morphology was observed by naked eyes. Cell morphology was visualized by light microscopy (CX21 microscope; Olympus, Japan). The Gram staining was carried out with a Gram staining kit (bioMérieux, Marcy l'Etoile, France). The cultivation temperature was 30°C unless indicated otherwise. Cell motility was tested in LB medium with 0.4% agar. Growth at various temperatures (20, 25, 32, 35, 37, 41, and 44°C) was tested in 5-ml aliquots of BHI broth dispensed into tubes (16-mm inner diameter) as described previously (5). Salt tolerance tests at different NaCl concentrations (0% to 10%, wt/vol, in increments of 1.0%) were performed in tryptic soy broth (TSB; Hopebio, Qingdao, China) after incubation for 2 days. Growth at pH 4.0 to 11.0 (at intervals of 1 pH unit, adjusted by adding HCl or NaOH) was examined in TSB for 2 days. The anaerobic growth was examined on a BHI agar plate, which was placed in an anaerobic bag (bioMérieux) at 30°C for 7 day (26). Aerobic acid production from glucose and gelatin hydrolysis was performed using the API 20NE system (bioMérieux), and the results were observed after 48 h. Hemolysis of sheep blood and utilization of citrate (Simmons’) were examined according to methods described previously (5). The characteristics for the assimilation of the other carbon sources were determined using the basal mineral medium (54) supplemented with 0.1% (wt/vol) carbon source as described previously (5).

Curation of all available Acinetobacter genomes for precise species identification.

All genome sequences labeled as Acinetobacter species in GenBank (n = 5,997, accessed by 1 August 2020) were retrieved. The assemblies, completeness, contamination, and heterogeneity of the genomes were evaluated using QUAST v5.0.2 (55) and CheckM v1.1.3 (45). Genome assemblies were discarded due to low quality defined by >300 contigs, a <50-kb N50 value, <90% genome completeness, genome contamination indicated by ≥2 in CheckM, or none-zero genome heterogeneity value for individual genomes. ANI and isDDH values between each of the genomes and type strains of Acinetobacter genomes were calculated, using the fastANI v1.32 (46) and genome-to-genome distance calculator (formula 2) (47), respectively. A ≥96% ANI (31) or ≥70.0% isDDH (31, 47) was used as the cutoff to define a bacterial species. AAI was calculated between each pair of genome sequences using CompareM v0.1.2 (56) with the recommended parameters.

Data availability.

The nearly complete 16S rRNA gene sequences, partial rpoB sequences, and the whole-genome shotgun projects of strains A. tianfuensis WCHAc060012T and A. rongchengensis WCHAc060115T have been deposited at DDBJ/ENA/GenBank under accession no. MK796537, MK796539, MK805088, MK805090, RAXV00000000, and RAXT00000000 (Table 2).

ACKNOWLEDGMENTS

We are grateful for Alexandr Nemec and his group at the National Institute of Public Health, Czech Republic for standardizing phenotypic characterizations and the helpful discussion.

The work was supported by grants from the National Natural Science Foundation of China (project no. 81861138055) and West China Hospital of Sichuan University (1.3.5 project for disciplines of excellence, project no. ZYYC08006).

We declare that we have no conflict of interest.

Contributor Information

Zhiyong Zong, Email: zongzhiy@scu.edu.cn.

Rup Lal, University of Delhi.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FIG S1

Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences (1,352 bp) of WCHAc060012T, WCHAc060115T, and type strains of Acinetobacter species with validly published names. The sequence of Moraxella lacunata ATCC 17967T (GenBank accession no. AF005160) was used as the outgroup. Bootstrap values (≥50%) after 1,000 resamplings are indicated at branch nodes. Shown in parentheses are the DDBJ/ENA/GenBank accession no. for 16S rRNA gene sequences or whole-genome sequences. Bar, 0.2 substitutions per nucleotide position. Download FIG S1, PDF file, 2.6 MB (2.7MB, pdf) .

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FIG S2

Maximum likelihood phylogenetic tree based on partial rpoB (861 bp) gene sequences of WCHAc060012T, WCHAc060115T, and type strains of Acinetobacter species with validly published names. Evolutionary distances were computed using Kimura’s two-parameter model. Moraxella lacunata ATCC 17967T was used as the outgroup. Bootstrap values ≥ 50% based on 1,000 resamplings are shown. Shown in parentheses are the DDBJ/ENA/GenBank accession no. for rpoB gene sequences or whole-genome sequences. Bar, 0.2 substitutions per nucleotide position. Download FIG S2, PDF file, 2.3 MB (2.3MB, pdf) .

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DATA SET S1

Phenotypic properties of A. tianfuensis sp. nov., A. rongchengensis sp. nov. and the Acinetobacter species with validly published names. Download Data Set S1, XLSX file, 0.03 MB (30.3KB, xlsx) .

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TABLE S1

Antimicrobial resistance genes of A. tianfuensis WCHAc060012T and A. rongchengensis WCHAc060115T. Download Table S1, PDF file, 0.1 MB (71.7KB, pdf) .

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TABLE S2

The characteristics of strains of A. pullorum and A. portensis. Download Table S2, PDF file, 0.1 MB (143.5KB, pdf) .

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TABLE S3

The 55 Acinetobacter genome sequences with misidentified species in NCBI. Download Table S3, PDF file, 0.1 MB (92.7KB, pdf) .

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DATA SET S2

The 3,956 Acinetobacter genome sequences available in GenBank (accessed by 1 August 2020). Download Data Set S2, XLSX file, 0.4 MB (427.5KB, xlsx) .

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DATA SET S3

The potential tentative species designation. Download Data Set S3, XLSX file, 0.02 MB (16.1KB, xlsx) .

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DATA SET S4

Average nucleotide identity between type strains of Acinetobacter species and reference strains of Acinetobacter taxa without species names. Download Data Set S4, XLSX file, 0.1 MB (124.3KB, xlsx) .

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DATA SET S5

Average amino acid identity between type strains of Acinetobacter species and reference strains of Acinetobacter taxa without species names. Download Data Set S5, XLSX file, 0.1 MB (129.7KB, xlsx) .

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Data Availability Statement

The nearly complete 16S rRNA gene sequences, partial rpoB sequences, and the whole-genome shotgun projects of strains A. tianfuensis WCHAc060012T and A. rongchengensis WCHAc060115T have been deposited at DDBJ/ENA/GenBank under accession no. MK796537, MK796539, MK805088, MK805090, RAXV00000000, and RAXT00000000 (Table 2).


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