TAXONOMIC DESCRIPTION Meng et al., Int J Syst Evol Microbiol 2017;67:1873–1879 DOI 10.1099/ijsem.0.001884
Actinomyces liubingyangii sp. nov. isolated from the vulture Gypaetus barbatus Xiangli Meng,1† Shan Lu,1† Xin-He Lai,2† Yiting Wang,1,3† Yumeng Wen,1 Dong Jin,1 Jing Yang1 and Jianguo Xu1,4,*
Abstract Two strains (VUL4_1T and VUL4_2) of Gram-staining-positive, catalase-negative, non-spore-forming short rods were isolated from rectal swabs of Old World vultures (Gypaetus barbatus) in the Tibet-Qinghai Plateau, China. Analysis of morphological characteristics and biochemical tests indicated that the two strains closely resembled each other but were distinct from other species of the genus Actinomyces previously described. Based on the results of 16S rRNA gene sequence comparison and genome analysis, strains were determined to be members of the genus Actinomyces, closely related to the type strains of Actinomyces marimammalium (96.4 % 16S rRNA gene sequence similarity), Actinomyces hongkongensis (92.4 %), Actinomyces hordeovulneris (92.3 %) and Actinomycesnasicola (92.2 %), respectively. Optimal growth conditions were 37 C, pH 6–7, with 1 % (w/v) NaCl. Strain VUL4_1T contained C18 : 1!9c and C16 : 0 as the major cellular fatty acids and diphosphatidylglycerol as the major component of the polar lipids. The genomic DNA G+C content of VUL4_1T was 54.9 mol%. Strain VUL4_1T showed less than 70 % DNA–DNA relatedness with other species of the genus Actinomyces, further supporting strain VUL4_1T as a representative of a novel species. Based on the phenotypic data and phylogenetic inference, a novel species, Actinomyces liubingyangii sp. nov., is proposed with VUL4_1T (=CGMCC 4.7370T=DSM 104050T) as the type strain.
As one of the largest Gram-staining-positive genera within the class Actinobacteria, the genus Actinomyces is a heterogeneous group of rod-shaped organisms with a high DNA G+C content that are non-spore-forming, non-motile and facultatively anaerobic [1]. Classification and identification of species of the genus Actinomyces has greatly improved in recent years, leading to the isolation of several novel species from human and animal specimens with pathogenic potential [2–6]. Animals, especially wild animals, have demonstrated their importance as reservoirs of many human pathogens [7–11]. The Old World vultures may carry and spread infectious pathogens, since they feed on carcasses of dead animals and participate in the human sky burials in the region studied, some involving people who died from communicable diseases [12–16]. The intestinal microbiome of these carrion
scavengers holds special significance from both evolutionary and human health perspectives. To this end, rectal swabs were collected from several vultures (Gypaetus barbatus) at Qinghai-Tibet plateau and dispensed in 2 ml sterile tubes containing Luria–Bertani (LB) medium with 20 % glycerol. These samples were subjected to analysis of their intestinal microbial composition by dilutions (10 1 to 10 2) and incubation aerobically on BHI-sheep blood agar (BD, Becton). Two novel strains, VUL4_1T and VUL4_2, were isolated from the plates, and they were phylogenetically and phenotypically closely related to members of the genus Actinomyces with differing and distinct features from recognized species. This paper describes this novel species of the genus Actinomyces. Gram-staining and morphological features were tested with cells grown on BHI-sheep blood agar plates at 37 C for 48 h
Author affiliations: 1State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Changping, Beijing 102206, PR China; 2Department of Pediatrics & Institute of Translational Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, PR China; 3Institute for Immunization and Prevention, Beijing Center for Diseases Prevention and Control, Beijing, PR China; 4Shanghai Institute for Emerging and Remerging Infectious Diseases, Shanghai Public Health Clinical Center, Jinshan, Shanghai, PR China. *Correspondence: Jianguo Xu,
[email protected] Keywords: Actinomyces liubingyangii; Qinghai-Tibet Plateau; vulture. †These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains VUL4_1T and VUL4_2 are KY268292 and KY268291, respectively. The GenBank/EMBL/DDBJ accession numbers for the genome sequences of strain VUL4_1T, VUL4_2, A. marimammalium CCUG 41710T, A. nasicola CCUG 46092T and A. hordeovulneris DSM 20732T are MQSU00000000, MQSV00000000, MPDM00000000, MQVR00000000 and MQVS00000000, respectively. One supplementary table and five supplementary figures are available with the online Supplementary Material. 001884 ã 2017 IUMS
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Meng et al., Int J Syst Evol Microbiol 2017;67:1873–1879
with incubation in an air plus 5 % CO2 atmosphere. Gramstaining was performed using a Gram stain kit (bioMerieux) according to the manufacturer’s instructions, and cell morphology was examined with light and transmission electron microscopes (HT7700, Hitachi). Growth conditions of the two novel strains were tested at 10 different temperatures (4, 10, 20, 25, 30, 37, 40, 45, 50 and 55 C) with media of eight different pH values (pH 4, 5, 6, 7, 8, 9, 10, 11) adjusted with HCl or NaOH (1 M), five different NaCl concentrations (0, 1, 2, 3, 4 %, w/v) and the following aeration conditions: anaerobic, microaerophilic (using anaeroGEN or microaerGEN bags, Thermo Scientific) and aerobic in the presence of 5 % CO2, respectively. Cell motility was tested by motility assays in soft agar plates as described previously [17]. The results from the above experiments showed that strains VUL4_1T and VUL4_2 were both Gram-stainingpositive, facultatively anaerobic, non-motile and grew well under 5 % CO2. The cells were short-rod-shaped (0.6– 1.00.8–1.2 µm) (Fig. S1, available in the online Supplementary Material), and their colonies were circular, greyish white, opaque and shiny at 37 C after 48 h of aerobic incubation with 5 % CO2. Optimum growth was achieved at 37 C, pH 6–7 and with 1 % (w/v) NaCl. Unless stated otherwise, all tests characterizing strain VUL4_1T and VUL4_2 were conducted under optimal growth conditions. The isolates were characterized biochemically by using API 50 CH strips (carbohydrate fermentation) combined with API 50 CHB/E medium, API ZYM (enzymic profiling), API Coryne (identification of coryneform bacteria) and the API Rapid ID 32 STREP system (identification of streptococcus) according to the manufacturer’s instructions (bioMerieux). The strains were cultured on BHI-sheep blood plates for 48 h in air plus 5 % CO2. Acid was produced from glycerol, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, maltose, lactose and potassium 5-ketogluconate (weakly); the strains could not produce acid from erythritol, D-arabinose, Larabinose, D-ribose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, salicin, cellobiose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, Dfucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate or methyl-b D-glucopyranoside, and did not produce acetoin (50 CH and Rapid ID 32 STREP). Positive activity was detected for alkaline phosphatase, leucine arylamidase, trypsin (weakly positive), acid phosphatase (weakly positive), naphthol-AS-BI-phosphohydrolase, bgalactosidase, a-glucosidase, b-glucosidase (only strain VUL4_2), N-acetyl-b-glucosaminidase, pyrazinamidase, alanyl-phenylalanine proline arylamidase, glycyl-tryptophan arylamidase and hydrolysis of hippurate; negative activity was found for catalase, lipase, valine arylamidase, cystine arylamidase, a-galactosidase, b-glucuronidase, b-glucosidase (only strain VUL4_1T), a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, arginine dihydrolase, pyroglutamic acid arylamidase, b-mannosidase and urease. (API ZYM, API Coryne and API Rapid ID 32 STREP).
The physiological characteristics of VUL4_1T and VUL4_2 were compared with their phylogenetically close relatives (Actinomyces marimammalium CCUG 41710T, Actinomyces hongkongensis DSM 15629T, Actinomyces hordeovulneris DSM 20732T and Actinomyces nasicola DSM 19116T). All the strains under comparison were examined at the same time, and the key results were summarized in Table 1. Although A. nasicola DSM 19116T only fermented starch weakly, the negative reactions for valine arylamidase and starch fermentation were the two defining features of VUL4_1T and VUL4_2, differentiating them from the other four species in Table 1. The results from the other three tests (negative for arginine dihydrolase, positive in fermenting Dxylose and hydrolysing hippurate) helped differentiate VUL4_1T and VUL4_2 from at least three other species, with slightly decreasing power of discrimination. Strain VUL4_1T could also be distinguished from A. marimammalium CCUG 41710T by characteristics of acid production from L-sorbose, D-mannose and D-galactose, and activity of a-glucosidase. The isolate differed from A. nasicola DSM 19116T also by the reactions of L-arabitol, potassium gluconate, L-fucose, cellobiose, D-galactose, b-glucosidase, amannosidase, alkaline phosphatase and acid phosphatase. It differed from A. hordeovulneris DSM 20732T by many more tests, such as acid production from melibiose, raffinose, cellobiose and glycerol, b-glucosidase, a-mannosidase, agalactosidase, pyroglutamic acid arylamidase, alkaline phosphatase, acid phosphatase, catalase, hydrolysis gelatin and a-galactosidase. VUL4_1T could be distinguished from A. hongkongensis DSM 15629T by the characteristics of acid production from melibiose, raffinose, L-fucose and D-mannose, b-galactosidase, N-acetyl-b-glucosaminidase and aglucosidase (Table 1). To identify the chemotaxonomic characteristics of the two novel strains, the cellular fatty acids and polar lipids of VUL4_1T and VUL4_2 were investigated. The celluar fatty acids were extracted according to the protocol of the Sherlock Microbial Identification System and detected with a gas chromatograph (HP6890; Hewlett Packard) [18]. The polar lipids were studied following previously described methods using two-dimensional TLC and staining with molybdatophosphoric acid [19, 20]. Isoprenoid quinone was investigated as described by Collins and Jones [21]. The major cellular fatty acids of strains VUL4_1T and VUL4_2 were very similar (31.2 % C16 : 0 and 32.3 % C18 : 1!9c for VUL4_1T vs 36.1 % C16 : 0 and 35.3 % C18 : 1!9c for VUL4_2). Fatty acid C18 : 0 was found in minor amounts. The complete fatty acids profiles of VUL4_1T, VUL4_2 and the four reference strains are presented in Table 2. MK-10 (H4) was the major respiratory quinone for both novel strains. Polar lipids were characterized with spray reagents specific for phosphate (molybdenum blue spray reagent), free amino groups (ninhydrin), sugars (a-naphthol) and quaternary nitrogen compounds (Dragendorff), and the nonspecific spray reagent (phosphomolybdic acid hydrate). The polar lipids profiles of strain VUL4_1T and VUL4_2 contained diphosphatidylglycerol as the major compound. There were moderate
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Meng et al., Int J Syst Evol Microbiol 2017;67:1873–1879
Table 1. Physiological characteristics that differentiate VUL4_1T and VUL4_2 from their closest phylogenetic relatives in the genus Actinomyces Strains: 1, VUL4_1T; 2, VUL4_2; 3, A. marimammalium CCUG 41710T; 4, A. nasicola DSM 19116T; 5, A. hordeovulneris DSM 20732T; 6, A. hongkongensis DSM 15629T. +, Positive; , negative; W, weakly positive; V, variable. Characteristic/test
1
2
3
4
5
6
Acid production (API 50 CH) L-Arabitol,
potassium gluconate
+
Melibiose, raffinose
+
L-Fucose
+
Starch
+
Cellobiose L-Sorbose
+ +
W
+
+
+
+
+
+
+
D-Mannose
+
+
D-Galactose
+
+
+
D-Xylose
+
+
+
Glycerol
+
+
+
+
+ W
Enzyme activity (API ZYM, Rapid ID 32 Strep) +
b-Glucosidase
+
a-Mannosidase b-Galactosidase, N-acetyl-b-glucosaminidase
+
+
+
Alkaline phosphatase, acid phosphatase
+
+
W
a-Glucosidase
+
+
+
+
+
+
+
a-Galactosidase, pyroglutamic acid arylamidase
+
Valine arylamidase
+
Arginine dihydrolase
V
+
+
+
+
+
+
+
+
API Coryne Catalase, hydrolysis gelatin, a-galactosidase Hydrolysis of hippurate
+ +
amounts of phosphatidylinositol mannoside and trace amounts of phosphatidylglycerol, two unidentified aminolipids and two unidentified polar lipids (Fig. S2). A highly similar polar lipid profile was detected in A. marimammalium CCUG 41710T but the presence of minor amounts of phosphatidylinositol, an unidentified phosphoglycolipid, a third unidentified polar lipid and two other unidentified aminolipids distinguished A. marimammalium CCUG 41710T from VUL4_1T and VUL4_2 (Fig. S2). The results of polar lipids profiling also supported the affiliation of the novel isolates with the genus Actinomyces. To investigate the phylogenetic relationships of the novel organisms with other existing species, the almost-complete 16S rRNA gene sequences of VUL4_1T and VUL4_2 were amplified with two universal bacterial primers, the forward primer 8F (5¢-AGAGTTTGATCMTGGCTCAG-3¢) and the reverse primer 1492R (5¢-GGYTACCTTGTTACGACTT-3¢) [22]. The 16S rRNA gene sequences of the novel strains were obtained from sequencing and compared with sequences in GenBank by using BLAST through the NCBI server. The 16S rRNA gene sequence alignment results indicated that VUL4_1T was 99.1 % similar to strain VUL4_2. Furthermore, strain VUL4_1T was closely related to A. marimammalium CCUG 41710T (96.4 % 16S rRNA gene sequence similarity), A. hongkongensis DSM 15629T (92.4 %), A. hordeovulneris DSM 20732T (92.3 %) and A. nasicola CCUG 46092T
+
+
(92.2 %), respectively. Its similarity level (below 98.7 %) in 16S rRNA gene sequence with other species of the genus Actinomyces demonstrated that VUL4_1T is very likely a representative of a novel species within the genus Actinomyces [23]. The 16S rRNA gene sequence phylogenetic trees were subsequently reconstructed with three different algorithms, the neighbour-joining algorithm [24], the maximum likelihood algorithm [25] and the maximum-parsimony algorithm [26] using MEGA 7 based on 1000 bootstrap replications. The evolutionary distances were computed using the Kimura 2parameter method [27]. Treeing analysis of 16S rRNA gene sequences showed that VUL4_1T was most closely related to A. marimammalium CCUG 41710T (Figs 1, S3 and S4). Phylogenetic analysis further demonstrated the evolutionary position of the unidentified bacterium within the genus Actinomyces. The chromosomal DNA of VUL4_1T and VUL4_2 was isolated with a Wizard Genomic DNA Purification kit (Promega) as per the manufacturer’s instructions and sequenced with an Illumina Hiseq 2500 by using a paired-end 125 bp sequencing strategy with the genomic DNA from A. marimammalium CCUG 41710T as the calibration reference in the sequencing and analysis. A total of more than 2 Mbp were obtained for VUL4_1T and assembled into 15 contigs in 10 scaffolds using SOAP de novo (version 2.04), which gave a coverage depth of approximately 100. Strain
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Meng et al., Int J Syst Evol Microbiol 2017;67:1873–1879
Table 2. Major cellular fatty acid contents of VUL4_1T, VUL4_2 and the type strains of closely related species in the genus Actinomyces Fatty acid (%)
VUL4_1T
VUL4_2
A. marimammalium
A. nasicola
A. hordeovulneris
A. hongkongensis
Saturated acids C12 : 0
1.1
TR
TR
TR
0.5
–
C14 : 0
6.4
2.5
2.0
6.7
22.1
4.1
TR
TR
TR
8.1
10.7
–
C16 : 0
31.2
36.1
31.5
39.9
42.5
45.9 –
anteiso-C15 : 0 C17 : 0
0.6
0.6
1.7
0.9
0.6
anteiso-C17 : 0
TR
TR
0.6
1.2
1.3
–
C18 : 0
9.4
10.4
10.1
6.1
4.1
10.2
Unsaturated acids C16 : 1!9c
6.0
1.6
0.6
TR
–
–
C17 : 1!8c
–
0.5
0.7
0.5
TR
–
C18 : 1!9c
32.3
35.3
24.7
13.9
4.5
24.6
C20 : 1!9c
–
TR
1.1
–
–
–
C20 : 4!6,9,12,15c
–
TR
–
–
0.8
–
3
1.0
1.2
1.1
1.3
0.8
1.8
4
–
TR
0.6
–
–
–
5
8.1
6.0
18.1
14.1
5.3
13.4
8
3.3
2.3
5.6
2.6
1.5
–
Summed features*
TR,
Trace level (
