DOI: 10.15468/cqp3na
title: Actinomyces qiguomingii sp. nov., isolated from the Pantholops hodgsonii

description: This dataset contains the digitized treatments in Plazi based on the original journal article Zhu, Wentao, Yang, Jing, Lu, Shan, Lai, Xin-He, Jin, Dong, Wang, Xiaoxia, Pu, Ji, Ren, Zhihong, Huang, Ying, Wu, Xiaomin, Zhang, Xiaoyan, Xu, Jianqing, Xu, Jianguo (2020): Actinomyces qiguomingii sp. nov., isolated from the Pantholops hodgsonii. International Journal of Systematic and Evolutionary Microbiology 70 (1): 58-64, DOI: 10.1099/ijsem.0.003709, URL: http://dx.doi.org/10.1099/ijsem.0.003709AbstractTwo previously undescribed, Gram-stain-positive, rod-shaped strains, 410 T and 553, were isolated from faeces of the Tibetan antelope (Pantholops hodgsonii) from the Tibet-Qinghai Plateau, PR China. The optimum growth conditions of the two novel strains were 1% (w/v) NaCl, 37 °C and pH 7. The end products from glucose fermentation included ethanol and lactic acid. Based on results of 16S rRNA gene sequence comparison and phylogenetic and phylogenomic analyses, strains 410 T and 553 were classified into the genus Actinomyces, and were closely related to Actinomyces ruminicola (97.6%), Actinomyces oricola (93.5%) and Actinomyces dentalis (90.8%). The genomic G+C content of strain 410 T was 67.4 mol%. Digital DNA–DNA hybridization values between strain 410 T and each of the closely related species were under 70%. The respiratory quinones were MK-10 (68%) and MK-9 (32%). The main cellular fatty acids of the isolates were C 16:0, followed by C 18:1 ω 9 c. The major polar lipids were diphosphatidylglycerol and phosphatidylinositol-mannoside. The whole-cell sugars contained rhamnose, ribose and glucose. The diagnostic amino acids of cell-wall peptidoglycan included alanine, glutamic acid, lysine and ornithine. The results of biochemical, chemotaxonomic and genotypic analyses revealed that the two novel strains represent a novel species of genus Actinomyces, for which the name Actinomyce s qiguomingii sp. nov. is proposed. The type strain is 410 T (= CGMCC 1.16361 T = DSM 106201 T).As of the beginning of 2019, the genus Actinomyces comprises 46 species (www.bacterio.net/a/actinomyces.html). Cells of the members in the genus are Gram-stain-positive, non-spore forming, non-motile, rod-shaped and anaerobic or facultatively anaerobic with high genomic G+C contents [1]. Most of the species are from animals and human clinical specimens, such as Actinomyces europaeus [2] and Actinomyces graevenitzii [3] from human clinical sources, and Actinomyces weissii [4] and Actinomyces marimammalium [5] from animal samples. In previous studies, we identified four novel species of the genus Actinomyces, including Actinomyces gaoshouyii from plateau pika [6] and three others from different vulture species [7 – 9].In the present study, we describe two strains isolated from faecal samples of two Tibetan antelopes (Pantholops hodgsonii) from different geographical sites in Qinghai, PR China (35° 59′ N 94° 03′ E and 35° 51′ N 94° 03′ E). The Tibetan antelope is endemic to the Qinghai-Tibet Plateau, where the altitude ranges from 4000 to 5000 meters [10, 11]. Faecal samples of Tibetan antelopes were obtained as described previously [12], diluted with sterile distilled water and mixed well. Brain heart infusion agar sheep blood plates (BHI–sheep blood agar plates) were evenly spread with the resultant supernatant and cultured at 37 °C in air plus 5% CO 2 for 5 days. Single white colonies that emerged on the plates were purified and subjected to further analyses.Author affiliations: 1 Shanghai Institute for Emerging and Re-emerging Infectious Diseases, Shanghai Public Health Clinical Center, Shanghai 201508, PR China; 2 State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing 102206, PR China; 3 School of Biology and Food Sciences, Shangqiu Normal University, Shangqiu, Henan 476000, PR China; 4 Shaanxi Institute of Zoology, Xi'an 710032, PR China.*Correspondence: Jianguo Xu,xujianguo@icdc.cnKeywords: Actinomyces qiguomingii sp. nov.; Tibetan antelope; Qinghai-Tibet Plateau.Abbreviations: ANI, average nucleotide identity; BHI, brain heart infusion; dDDH, digital DNA–DNA hybridization.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains 410 T and 553 are MG 650117 and MH188287, respectively. The GenBank/EMBL/DDBJ accession number for the complete genome sequence of strain 410 T is CP025228. The GenBank/EMBL/ DDBJ accession number for the draft genome sequences of strains 553 and Actinomyces oricola JCM 17565 T are PPPL00000000 and RYFV00000000, respectively.One supplementary table and four supplementary figures are available with the online version of this article.All microbiological characteristics of the two strains were collected from cells grown for 2 days at 37 °C in air plus 5% CO 2 on BHI-sheep blood agar plates. Gram-staining was performed using a Gram-staining kit in accordance with the manufacturer's instructions. Both light and transmission electron microscopy (HT7700, Hitachi) were applied to observe the cell morphology. The cells were Gram-stain-positive, nonmotile, rod-shaped, 0.3–0.5 µm wide and 2.1–2.8 µm long (Fig. S1, available in the online version of this article). The colonies on BHI-sheep blood agar after 72 h incubation in air plus 5% CO 2 were convex, circular, opaque, milky white and about 1 mm. BHI broth was used to determine the optimal growth conditions by monitoring OD 600 at 3 and 7 days. Growth conditions were tested at seven pH levels (from pH 4 to pH 10, at intervals of 1.0 pH unit, adjusted using NaOH or HCl) [13], five levels of temperatures (20, 28, 37, 42 and 50 °C) and five NaCl concentrations [0, 1, 2, 3 and 4% (w/v)]. The isolates grew at 28–42 °C, at pH 6.0–9.0 and at 0–3% (w/v) NaCl in air plus 5% CO 2. Optimum growth occurred at 37 °C, pH 7.0 and 1% (w/v) NaCl. The oxidase activity and catalase activity were negative when tested with 0.1% (w/v) tetramethyl- p -phenylenediamine and 3% (v/v) H 2 O 2 solution, respectively. The end products of glucose fermentation by strain 410 T, as determined following the procedure of An et al. [13], were composed of ethanol and lactic acid. Analyses of the composition of cellular fatty acids, isoprenoid quinones and polar lipids were performed using bacterial cells grown under the optimal growth conditions, unless otherwise indicated.The 16S rRNA gene was amplified using universal primers 27F and 1492R [14], the PCR products were gel purified and cloned into vector pMD-18 T for sequencing, and the almost full-length 16S rRNA gene sequences were used to determine the phylogenetic position of the isolates as described previously [15]. The 16S rRNA gene sequence similarity between the two isolates was 99.7%. Comparative analyses of 16S rRNA gene sequences showed that the isolates are closely related to Actinomyces ruminicola CGMCC 1.5030 T (97.6%) and Actinomyces oricola JCM 17565 T (93.5%). 16S rRNA gene sequence-based phylogenetic trees were subsequently established using the neighbour-joining [16], maximumlikelihood [17] and maximum-parsimony [18] algorithms using MEGA version X [19]. The grouping stabilities and evolutionary distances were computed by bootstrap analysis (1000 replications) and the Kimura two-parameter method, respectively [20]. The neighbour-joining phylogenetic tree showed that strains 410 T and 553 were positioned within the genus Actinomyces, forming a separate cluster with A. ruminicola CGMCC 1.5030 T, A. dentalis DSM 19115 T and A. oricola JCM 17565 T (Fig. 1). The taxonomic position of the isolates was similar to phylogenetic trees built using the maximum-likelihood (Fig. S2) and maximum-parsimony (Fig. S3) algorithms.Bacterial genomic DNA was extracted using a Wizard Genomic DNA Purification Kit according to the manufacturer’s instructions and purified by the ethanol precipitation method. The complete genome sequence of strain 410 T (CP025228) was sequenced on the PacBio platform and de novo as a circular chromosome by HGAP 3.0 [21]. Meanwhile, the draft genome sequences of strains 553 (PPPL00000000) and A. oricola CCUG 46090 T (RYFV00000000) were sequenced using Illumina apparatus and assembled using VELVET [22]. The circular genome of strain 410 T (3.99 Mb) was predicted by Prodigal software to contain 3389 coding genes, with an average length of 1000bp (data not shown). The tRNA-scan tool was used to predict the types and locations of RNAs, which suggested that the strain 410 T carried 9 rRNAs and 48 tRNAs in its genome [23]. The G+C content of the chromosomal DNA of strain 410 T was 67.4 mol %, within the range of those of the members in the genus Actinomyces.A phylogenomic tree was reconstructed with Bifidobacterium bifidum ATCC 29521 T as an outgroup using FastTree and visualized in Dendroscope 3 as described previously [12].Based on 0.4 protein sequence identity threshold, 178 core genes (supplement excel file) were extracted from the three genomes that we sequenced (strains 410 T, 553 and A. oricola JCM 17565 T) in this study and the 37 genomes of the genus Actinomyces already available from the NCBI GenBank database. The phylogenomic tree (Fig. 2) showed that our strains were closely related to A. ruminicola CGMCC 1.5030 T and A. dentalis DSM 19115 T.A DNA–DNA hybridization (DDH) value below 70% between two bacterial strains, the suggested threshold level [24], is considered to indicate different species. The digital DDH (dDDH) value between strains 410 T and 553 and their closely related species were calculated using a web-lab DDH model (http://ggdc.dsmz.de/). The dDDH value between strains 410 T and 553 was 91.4% (Table 1), whereas those between strain 410 T and each of the closely related species (A. ruminicola CGMCC 1.5030 T, A. dentalis DSM 19115 T and A. oricola JCM 17565 T) were 26.8, 22.5 and 21.5% (Table 1), respectively. The dDDH values between strain 410 T and other members of the genus Actinomyces were well below the 70% threshold (Table S1). The average nucleotide identity (ANI) values (Table 1) calculated using orthoANI web software [25] between strain 410 T and each of the closely related species were far below the 95–96% threshold [26], in contrast to the above threshold value (99.2%) between strain 410 T and 553.The biochemical properties of strains 410 T and 553 were tested using the commercially available API 50 CH strip combined with API 50 CHB/E medium, API Rapid ID 32A and the API ZYM system according to the manufacturer’s instructions (bioMérieux). Acid was produced from glycerol, D-arabinose, L-arabinose, D-ribose, D-xylose, D-galactose,D-glucose,D-fructose,D-mannose,L-sorbose, L-rhamnose, inositol, D-mannitol, D-sorbitol, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, potassium 5-ketogluconate (553 only) and potassium gluconate (API 50 CH). Enzymatic activity was positive for β -galactosidase, α -glucosidase, β -glucosidase, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase, serine arylamidase (weak), alkaline phosphatase, valine arylamidase, acid phosphatase and naphthol-AS-BIphosphohydrolase (API Rapid ID 32A and ZYM). The key characteristics that could distinctively differentiate the two novel strains from all their closely related species (A. ruminicola CGMCC 1.5030 T, A. dentalis DSM 19115 T and A. oricola JCM 17565 T) were alkaline phosphatase activity and acid production from D-mannitol, inulin and glycogen (Table 2). Other characteristics that differentiate strains 410 T and 553 from the closely related species are detailed in Table 2.Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between Actinomyces qiguomingii sp. nov. and the type strains of the genus Actinomyces. Solid circles indicate the nodes supported by both maximum-likelihood and maximum-parsimony analyses. Bootstrap values (70%) based on 1000 replicates are shown at branch nodes. Bar, 0.020 changes per nucleotide position.Fig. 2. Neighbour-joining phylogenomic tree of Actinomyces qiguomingii sp. nov. and other available strains of the most closely related species based on 178 core genes from genome sequences. Numbers on the tree indicate each split in the tree with support values from the Shimodaira–Hasegawa test [34] calculated for 1000 resamples. Bifidobacterium bifidum ATCC 29521 T was used as the outgroup.Table 1. Digital DNA–DNA hybridization (lower diagonal) and average nucleotide identity (upper diagonal) values between Actinomyces qiguomingii sp. nov. and the closely related species of the genus Actinomyces Strains: 1, 410T; 2, 553; 3, Actinomyces ruminicola CGMCC 1.5030T; 4, Actinomyces dentalis DSM 19115T; 5, Actinomyces oricola JCM 17565T. Digital DNA–DNA hybridization results are percentages and expressed based on Formula 2: sum of all identities found in high-scoring segment pairs (HSPs) divided by overall HSP length. Formula 2 is independent of genome length and is thus more robust when applied to incomplete draft genomes [24].Strains 1 2 3 4 5 1 100 (100) 99.2 83.8 78.5 75.5 2 91.4 100 (100) 84.1 78.0 75.5 3 26.8 26.9 100 (100) 71.0 64.9 4 22.5 22.6 22.0 100 (100) 78.3 5 21.5 21.4 21.7 22.2 100 (100)Table 2. Characteristics that differentiate Actinomyces qiguomingii sp. nov. from the closely related species of the genus Actinomyces Strains: 1, 410T; 2, 553; 3, Actinomyces ruminicola CGMCC 1.5030T; 4, Actinomyces dentalis DSM 19115T; 5, Actinomyces oricola JCM 17565T. −, Negative; +, positive; W, weak.Characteristic/test 1 2 3 4 5 API 50CH: Glycerol + + + − − L-Arabinose + + + − − D-Glucose + + + + − D-Mannose + + + + − Inositol + + + − − D-Mannitol + + − − − D-sorbitol + + + − − Sucrose + + + + − Inulin + + − − − Melezitose + + + − − Starch + + + − − Glycogen + + − − − Gentiobiose + + + + − D-Arabitol − − + − − Potassium 5-ketogluconate − + + − − API Rapid ID 32A and ZYM: Arginine arylamidase + + + + − Tyrosine arylamidase + + + − + Alanine arylamidase W + + + − Alkaline phosphatase + + − − − Esterase (C4) − − − + − Valine arylamidase + + − + − β -Galactosidase + + + + −The polar lipids were investigated as described previously [27, 28]. The cellular fatty acids were extracted according to the Sherlock Microbial Identification System [29]. Isoprenoid quinones of strain 410 T were analysed by reversed-phase high power liquid chromatography [30, 31]. The polar lipids of strain 410 T comprised diphosphatidylglycerol, phosphatidylinositol-mannoside, phosphatidylglycerol and an unidentified polar lipid (Fig. S4). The major polar lipids were diphosphatidylglycerol and phosphatidylinositolmannoside, which were similar to those of the genus Actinomyces. The complete cellular fatty acid profile (&gt;1%) of strain 410 T contained C 12:0 (3.9%), C 14:0 (4.7%), C 16:1 ω 9 c (2.1%), C 16:0 (47.4%), C 18:2 ω6,9 c / anteiso -C 18:0 (3.0%), C 18:1 ω 9 c (29.2%) and C 18:0 (6.0%). The complete cellular fatty acid profile (&gt;1%) of strain 553 contained C 12:0 (3.0%), C 14:0 (4.1%), C 16:1 ω 9 c (2.1%), C 16:0 (44.7%), C 18:2 ω 6,9 c / anteiso -C 18:0 (2.9%), C 18:1 ω 9 c (34.3%) and C 18:0 (4.5%). The major cellular fatty acids of strains 410 T and 553 were C 16:0, followed by C 18:1 ω 9 c, which were the same as those of closely related species. Isoprenoid quinones of strain 410 T were MK-10 (68%) and MK-9 (32%), which were highly similar to A. ruminicola CGMCC 1.5030 T [MK-10 (70%) and MK-9 (30%)] with slight differences in abundance [13]. The whole-cell sugars of strain 410 T, analysed as in Schleifer [32], contained rhamnose, ribose and glucose. The diagnostic amino acids of cell-wall peptidoglycan of strain 410 T included alanine, glutamic acid, lysine and ornithine, but not diaminopimelic acid [33].Based on the results of biochemical, chemotaxonomic and genotypic analyses, we propose the name Actinomyces qiguomingii sp. nov. for the novel bacteria, with 410 T as the type strain.