Chryseobacterium chengduensis sp. nov. isolated from the air of captive giant panda enclosures in Chengdu, China#

Chryseobacterium chengduensis sp. nov. isolated from the air of captive giant panda enclosures in Chengdu, China # # 分离自成都大熊猫兽舍空气中的一株金黄杆菌属新种的鉴定 1 first-author 1 1 1 1 1 1 1 2 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yanqigui@126.com †E-mail:yanqigui@126.com yanqigui@126.com 3 College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China College of Food Science, Sichuan Agricultural University, Yaan 625014, China College of Food Science, Sichuan Agricultural University, Yaan 625014, China Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China A Gram-negative, aerobic, non-motile, rod-shaped bacterial strain, designated 25-1 T , was isolated from the air inside giant panda enclosures at the Chengdu Research Base of Giant Panda Breeding, China. Strain 25-1 T grew optimally at pH 7.0–8.0, at 28–30 °C and in the presence of NaCl concentrations from 0.0% to 0.5 %. 16S rRNA gene sequence analysis indicated that strain 25-1 T belongs to the genus Chryseobacterium within the family Flavobacteriaceae and is related most closely to C . carnis G81 T (96.4% similarity), C. lathyri RBA2-6 T (95.8% similarity), and C. zeae JM1085 T (95.8% similarity). Its genomic DNA G+C molar composition was 36.2%. The major cellular fatty acids were iso -C 15:0 (44.0%), iso -C 17:0 3OH (19.8%) and C 16:1 ω7c/ 16:1 ω6c (12.7%). The only isoprenoid quinone was menaquinone 6 (MK-6). The major polar lipids were phosphatidylethanolamine, two unidentified amino lipids and two unidentified lipids. The DNA–DNA relatedness between strain 25-1 T and C. lathyri RBA2-6 T was 38%. Phenotypic, genotypic, and phylogenetic characteristics indicated that strain 25-1 T is a novel member of the genus Chryseobacterium , for which the name C. chengduensis sp. nov. is proposed. The type strain is 25-1 T (CCTCC AB2015133 T =DSM 100396 T ). 目的 2 鉴定菌株25-1 T 是否是金黄杆菌属的一个新种。 创新点 2 首次从空气中分离到金黄杆菌属的新种。 方法 2 革兰氏染色镜检；磷钨酸染色然后透射电镜观察菌株25-1 T 形态结构；全自动生理生化鉴定系统（Phoenix™-100）与传统生理生化反应管相结合；H890气象色谱仪进行脂肪酸组分分析；薄板双相层析进行极性脂组分分析；反相高压液相色谱分析法进行呼吸琨组分分析；熔解温度法检测G+C摩尔含量；16S rRNA序列测定及系统发生分析。 结论 2 根据传统特征分类研究结果（形态特征、培养特性和生理生化特征）、化学分类研究结果（脂肪酸组分、极性脂组分和呼吸琨组分）和遗传特征分类研究结果（G+C摩尔含量、DNA同源性测定和16S rRNA序列测定及系统发生分析）得出菌株25-1 T 为金黄杆菌属的一个新种。 Chryseobacterium chengduensis Giant panda 16S rRNA sequencing Phylogenetic analysis Strain 25-1 T 成都源金黄杆菌 大熊猫 16S rRNA测序 系统发育树分析 菌株25-1 T Introduction 1 The genus Chryseobacterium was first described by Vandamme et al. ( 1994 1994 ) and assigned to the family Flavobacteriaceae of the phylum Bacteroidetes. Descriptions of novel Chryseobacterium species have been growing rapidly in recent years. Currently, the genus contains over 90 species with valid published names ( http://www.bacterio.net/chryseobacterium.html http://www.bacterio.net/chryseobacterium.html ). Chryseobacterium strains are widely distributed in aquatic and soil environments, plant rhizospheres, sediments, and food sources (Cho et al., 2010 2010 ; Park et al., 2013 2013 ; Loch and Faisal, 2014 2014 ; Kämpfer et al., 2014a 2014a ; 2014b 2014b ; 2015a 2015a ). However, to the best of our knowledge, no airborne representatives of the genus have yet been described. Some Chryseobacterium strains are significant as novel sources of bioactive compounds, such as antioxidants, prebiotics, or as sulfobacin and protease producers (Chaudhari et al., 2009 2009 ; Wang et al., 2011 2011 ; Kim H.S. et al., 2012 2012 ). Moreover, some species, including C. indologenes , C . oranimense , and C . gleum , are opportunistic human pathogens implicated in nosocomial infections (Monteen et al., 2013 2013 ; Lo and Chang, 2014 2014 ; Nemli et al., 2015 2015 ; Sharma et al., 2015 2015 ). In veterinary medicine, no relevant Chryseobacterium species have been identified as a pathogen of domestic animals; nevertheless, numerous fish-associated species have been isolated from skin and muscle ulcerations, gill hemorrhage and hyperplasia, and from fish showing general signs of septicemia (Ilardi et al., 2009 2009 ; Loch and Faisal, 2014 2014 ). Cells are Gram-negative, strictly aerobic, non-motile, non-spore-forming, rods. They are pigmented yellow by flexirubin-type pigments with menaquinone 6 (MK-6) as the predominant respiratory quinone, branched-chain fatty acids ( iso -C 15:0 , iso -C 17:0 3-OH, and iso -C 17:1 (9c) as the major fatty acids and a DNA G+C content in the range from 29% to 39% (Kämpfer et al., 2009 2009 ; Bernardet et al., 2010 2010 ; Yang et al., 2015 2015 ). In this study, we investigated the cultivable bacterial community in the air of giant panda enclosures at the Chengdu Research Base of Giant Panda Breeding in Sichuan Province, located in south-western China. Based on differences in colony morphology, 28 pure cultures were selected for 16S rRNA gene sequencing and phylogenetic analysis. Species included members of the genera Micrococcus , Chryseobacterium , Leuconostoc , Staphylococcus , Pseudomonas , Kocuria , Bacillus , Exiguobacterium , Acinetobacter , Escherichia , Rothia , and Dietzia . The strain designated 25-1 T was characterized using a polyphasic taxonomy approach, including evaluation of its morphological, biochemical, and phylogenetic characteristics. Unfortunately, C . carnis G81 T and C . zeae JM1085 T had not been released from culture collections at the time of these investigations and so were not included as reference strains. Therefore, all tests were performed on the new isolate and on C. lathyri RBA2-6 T , which was acquired from the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC). The data obtained revealed that strain 25-1 T should be assigned to the genus Chryseobacterium as the type strain of a novel species. Materials and methods 1 Culture conditions and phenotypic characteristics 2 Strain 25-1 T was isolated from the cultivable bacterial community in the air of a giant panda enclosure by exposing a petri dish containing tryptic soy agar (TSA, Difco, Leeuwarden, the Netherlands) medium for 15 min. For further analysis, strain 25-1 T was cultivated on Luria-Bertani (LB) agar (Difco) at 30 °C. The presence of flexirubin type pigments was investigated using a 20% (0.2 g/ml) KOH solution according to the study of Bernardet et al. ( 2002 2002 ). Gram staining was determined using the non-staining method described by Buck ( 1982 1982 ). Cellular morphology, motility, and other physiological characteristics were evaluated as previously described (Wen et al., 2016 2016 ). Cellular morphology was observed by light microscopy (Olympus; magnification 61 000×) and cell size was determined by transmission electron microscopy (H-600-A2; Hitachi, Tokyo, Japan) using cells from an exponentially growing culture. Motility tests were performed using LB broth with 0.3% (3 g/L) agar. Growth temperatures (4, 10, 15, 20, 25, 28, 30, 37, 40, 45, and 50 °C) and pH (2.0–10.0, at intervals of 1.0 pH unit) were monitored during 7 d of incubation in LB broth as described by Xu and Wu ( 2005 2005 ). NaCl tolerance was tested in LN medium (LB without NaCl) supplemented with 0%, 0.5%, 1.0%–5.0% (at intervals of 1%) (1%=0.01 g/ml) NaCl during 7 d of incubation. Anaerobic growth was investigated by incubation in an anaerobic chamber (Mitsubishi Gas Chemical, Tokyo, Japan) at 30 °C for 7 d on LB agar. Biochemical characteristics and microbial sensitivity test 2 A number of key characteristics were tested using conventional procedures, as described by Smibert and Krieg ( 1994 1994 ) and Skerman ( 1967 1967 ), i.e., the production of catalase, oxidase, hydrogen sulphide and indole, and hydrolysis of Tween 80, starch, and gelatin. Some strain 25-1 T and C. lathyri RBA2-6 T biochemical reactions were detected using a bacterial biochemical trace kit (Hangzhou Microbial Reagent Co., Ltd., Hangzhou, China), which included the following substances: (-galactosidase, arginine decarboxylase, ornithine decarboxylase, nitrate reduction, mannose, adipic acid, arabinose, trehalose, cellobiose, lactose, salicin, and acetamide. The additional biochemical and physiological properties of strain 25-1 T and C. lathyri RBA2-6 T were determined using the BD Phoenix™-100 automated microbiology system (Becton Dickinson, New Jersey, USA), according to the manufacturer’s instructions. The biological principles of the Analytic Products INC (API) and Phoenix systems are similar (Wen et al., 2016 2016 ), but the Phoenix system is automated and can handle a higher number of tests. Each negative identification (NID) panel contains two fluorescent positive control wells and 45 substrates (O'Hara, 2006 2006 ). The sensitivity of strain 25-1 T to various antibiotics was determined as previously described (Wen et al., 2016 2016 ), by spreading bacterial suspensions on LB agar plates and applying filter paper discs containing the following antibiotics (μg per disc): vancomycin (30), sulfamethoxazole (100), tetracycline (30), lincomycin (10), spectinomycin (100), kanamycin (30), furazolidone (100), streptomycin (10), erythromycin (15), trimesulf (25), ciprofloxacin (5), rifampicin (5), ampicillin (10), florfenicol (30), penicillin (10), gentamicin (10), amikacin (30), cephalothin (30), ceftazidime (30), doxycycline (30), cefotaxime (30), and enrofloxacin (5) (all obtained from Hangzhou Microbial Reagents). The strain was incubated in the presence of the antibiotics at 30 °C for 24 h. 16S rRNA sequencing and phylogenetic analysis 2 The 16S rRNA gene of strain 25-1 T was amplified by polymerase chain reaction (PCR) using two universal primers as described previously (Greisen et al., 1994 1994 ), and the amplification products were sequenced by Invitrogen Biotechnology (Shanghai, China). The sequence was compared with sequences available in the EzTaxon Server ( http://eztaxon-e http://eztaxon-e . ezbiocloud.net; Kim O.S. et al., 2012 2012 ) and GenBank database ( http://blast.ncbi.nlm.nih.gov/Blast.cgi http://blast.ncbi.nlm.nih.gov/Blast.cgi ). The sequences of strain 25-1 T and the type strains of published Chryseobacterium species were aligned using the Clustal_X program (Thompson et al., 1997 1997 ). Phylogenetic analysis was performed using the MEGA 6.0 offline software (Tamura et al., 2013 2013 ) and PHYML online web server (Guindon et al., 2005 2005 ). Phylogenetic trees were constructed using the neighbor-joining, maximum-likelihood (Felsenstein, 1981 1981 ), and maximum-parsimony (Kluge and Farris, 1969 1969 ) methods in the MEGA 6.0 program, with bootstrap analysis based on 1000 replicates (Felsenstein, 1985 1985 ). Chemotaxonomic and genomic analyses 2 The DNA G+C composition of strain 25-1 T was determined using the thermal denaturation method (Mandel and Marmur, 1968 1968 ) with Escherichia coli K-12 as a control. Genomic DNA was extracted and purified using conventional procedures (Sambrook and Russell, 2001 2001 ). Polar lipids of strain 25-1 T were identified by two-dimensional thin-layer chromatography (TLC) according to the protocols of Tindall ( 1990 1990 ). The respiratory quinones of strain 25-1 T were extracted and analyzed by high performance liquid chromatography (HPLC) as described by Xie and Yokota ( 2003 2003 ), using C. takakiae CGMCC1.13488 T as a reference strain. The whole cell fatty acids of strains 25-1 T and C. lathyri RBA2-6 T grown on LB agar at 30 °C for 48 h were analyzed using the Sherlock microbial identification system (MIDI) and identified using the MIDI software package Version 6.0 based on the TSBA6 6.00 database. The four above-mentioned analyses were performed at the China General Microbiological Culture Collection Center (CGMCC). Genomic DNA–DNA hybridizations were carried out between strain 25-1 T and C. lathyri RBA2-6 T using the fluorometric method (Ezaki et al., 1989 1989 ), at the Guangdong Microbiology Culture Center (GIMCC). Results and discussion 1 Morphological and physiological characteristics 2 Cells of strain 25-1 T were observed to be non-motile, strictly aerobic, Gram-stain-negative, and rod-shaped (0.44–0.48 μm wide and 0.88–0.92 μm long; Fig. S1 S1 ). Colonies on LB agar were non-transparent, yellow-pigmented, circular, and smooth with regular edges after 3 d of incubation at 30 °C. The non-fluorescent and non-diffusible yellow pigments belong to the flexirubin type. Cells grow in LN medium with 0%–2% NaCl with an optimum concentration of 0%–0.5%. Good growth occurs at 28–30 °C and no growth occurs below 10 °C or above 40 °C. Growth occurs at pH 6.0–9.0 and optimally at pH 7.0–8.0. The characteristics of strain 25-1 T and C. lathyri RBA2-6 T are shown in Tables 1 1 and 2 2 . Strain 25-1 T was able to use acetate, adonitol, and D-mannitol, but unable to use ornithine, sorbitol, or sucrose. In contrast to C. lathyri RBA2-6 T , strain 25-1 T was negative for L -glutamic acid-AMC, γ-glutamyl-NA, L -proline-NA, bis- p NP-phosphate ( p NP: p -nitropheno), p NP-(- D -glucoside, and esculin hydrolysis. 1: strain 25-1 T ; 2: strain C. lathyri RBA2-6 T ; –: negative; +: positive. Data are from this study unless otherwise stated Characteristics 1 2 Catalase activity + + Oxidase activity + + Production  Indole – –  H 2 S – –  β-Galactosidase – –  Arginine decarboxylase – +  Ornithine decarboxylase + – Degradation  Starch + +  Tween 80 + +  Gelatin – +  Nitrate reduction – – Assimilation  Mannose – +  Adipic acid – –  Arabinose – –  Trehalose – –  Cellobiose – –  Lactose – –  Salicin – –  Acetamide – – 1: strain 25-1 T ; 2: strain C. lathyri RBA2-6 T ; AMC: 7-amido-4-methylcoumarin; MU: methylumbelliferyl; p NP: p -nitropheno; –: negative; +: positive. Data are from this study unless otherwise stated Characteristics 1 2 Hydrolysis  Arginine-arginine-AMC + +  Glycine-proline-AMC + +  Glycine-AMC + +  Glutaryl-glycine-arginine-AMC + +   L -Arginine-AMC + +   L -Glutamic acid-AMC – +   L -Leucine-AMC + +   L -Phenylalanine-AMC + +   L -Proline-AMC – –   L -Pyroglutamic acid-AMC + –   L -Tryptophan-AMC + +  Lysine-alanine-AMC + +   L -Proline-NA – +   p NP-β- D -glucoside – +  4MU- N -acetyl-β-D-glucosaminide + +  Bis- p NP-phosphate – +  γ-Glutamyl-NA – +  Urea – –  Esculin – + Utilization D-Mannitol + +  Tiglic acid + +  β-Allose – –  β-Gentiobiose – –  α-Ketoglutaric acid + +  Dextrose – – D-Fructose – – D-Galactose – – D-Gluconic acid – – D-Melibiose – –  Sorbitol – –  Sucrose – –  Acetate + +  Adonitol + +  Citrate + +  Galacturonic acid – – L-Arabinose – –  Malonate + + L-Rhamnose – –  Methyl-β-glucoside – –  Maltulose – – N -acetyl-galactosamine – – N -acetyl-glucosamine – –  Ornithine – – Resistance  Polymyxin B + +  Colistin + + Phylogenetic analysis 2 The 16S rRNA gene sequence (1401 bp) of strain 25-1 T was obtained (GenBank accession number KP966546). Strain 25-1 T showed less than 96.4% sequence similarity to the type strains of all recognized species in the genus Chryseobacterium . The highest 16S rRNA sequence similarity was found with C. carnis NCTC 13525 T (96.4%) (Holmes et al., 2013 2013 ), followed by C. lathyri RBA2-6 T (95.8%), C. zeae JM1085 T (95.8%) (Kämpfer et al., 2014b 2014b ), C. shigense GUM kaji T (95.8%) (Shimomura et al., 2005 2005 ), C. gwangjuense THG A-18 T (95.7%) (Park et al., 2013 2013 ), and C. carnipullorum 9-R23581 T (95.7%) (Charimba et al., 2013 2013 ), as determined using the EzTaxon server 2.1. Strain 25-1 T was included in a cluster containing the type strains of C. carnis and C. chaponense , forming a distinct phylogenetic lineage within the genus Chryseobacterium in the neighbor-joining phylogenetic tree (Fig. 1 1 ). The phylogenetic position was also supported by the maximum-parsimony and maximum-likelihood trees. GenBank accession numbers are shown in parentheses. Numbers indicate percentages of occurrence of branch points in 1000 bootstrapped trees. The bar is 0.5% in the legend and 0.005 on the tree Chemotaxonomic and genomic characteristics 2 The cellular fatty acids of strain 25-1 T and C. lathyri RBA2-6 T analyzed under the same conditions are shown in Table 3 3 . The predominant fatty acids of strain 25-1 T (≥5%) were iso -C 15:0 (44.0%), iso -C 17:0 3OH (19.8%), summed feature 3 (C 16:1 (7c and/or C 16:1 (6c, 12.7%) and summed feature 9 (10-methyl C 16:0 and/or iso -C 17:1 (9c, 7.8%). Minor amounts of anteiso -C 15:0 (1.4%), iso -C 16:0 (1.8%), iso -C 15:0 3OH (3.9%), iso -C 16:0 3OH (2.0%), and C 16:0 3OH (1.2%) were also detected. The presences of major fatty acids, namely iso -C 15:0 , iso -C 17:0 3OH and iso -C 17:1 (9c, are in accordance with the placement of strain 25-1 T in the genus Chryseobacterium (Li and Zhu, 2012 2012 ; Kämpfer et al., 2015b 2015b ). However, strain 25-1 T could be readily distinguished from C. lathyri by the presence of a high amount of iso -C 17:0 3OH and a significantly lower amount of summed feature 9. The discrepancies noted in the fatty acid composition of C. lathyri RBA2-6 T as determined in this study compared to the data reported in the original description (Cho et al., 2010 2010 ) may be due to differences in the fatty acid extraction methods, types of gas chromatography, or the culture media used. The isoprenoid quinone of strain 25-1 T was MK-6, which is characteristic of all members of the family Flavobacteriaceae (Kämpfer et al., 2009 2009 ). The polar lipids of strain 25-1 T were phosphatidylethanolamine (PE), two unidentified amino lipids and two unidentified lipids (Fig. 2 2 ), which is in line with other recognized species of the genus Chryseobacterium (Kämpfer et al., 2015a 2015a ). The DNA G+C molar content of strain 25-1 T was 36.2%. This value is within the range reported for Chryseobacterium species (Bernardet et al., 2010 2010 ; Montero-Calasanz et al., 2014 2014 ). The mean DNA–DNA relatedness between strain 25-1 T and C. lathyri RBA2-6 T was 38%. This is clearly far below the 70% threshold value that is generally used for prokaryotic species delineation (Stackebrandt et al., 2002 2002 ). Strain 1: C. chengduensis sp. nov. 25-1 T ; Strain 2: C. lathyri RBA2-6 T . All data are from this study. Fatty acids that represented ˂1.0% of the total in both strains are not shown. ND: not detected; TR: traces (i.e. < 1.0%). Summed features are groups of two or three fatty acids that cannot be separated by the Microbial Identification System. Summed feature 3 consisted C 16:1 (7c and/or C 16:1 (6c; summed feature 9 consisted of 10-methyl C 16:0 and/or iso -C 17:1 (9c 2 Fatty acid 2 Content (%) Strain 1 Strain 2 iso -C 15:0 44.0 53.5 anteiso -C 15:0 1.4 1.8 iso -C 15:1 F ND 1.4 iso -C 16:0 1.8 TR C 16:0 TR 1.8 iso -C 15:0 3OH 3.9 5.0 iso -C 16:0 3OH 2.0 1.5 C 16:0 3OH 1.2 ND iso -C 17:0 3OH 19.8 ND iso -C 17:0 ND 1.9 Summed feature 3 a a 12.7 10.7 Summed feature 9 a a 7.8 18.0 Ascending solvent system: 1st dimension: chloroform/methanol/water (65:25:4, v/v); 2nd dimension: chloroform/methanol/acetic acid/water (80:12:15:4, v/v). Molybdatophosphoric acid was applied for the detection of polar lipids. PE, phosphatidylethanolamine; AL1 and AL2, unidentified amino lipids; UL1 and UL2, unidentified lipids Conclusions 1 The physiological, chemotaxonomic, and phylogenetic analyses conducted in this study show that strain 25-1 T exhibits the main properties of the genus Chryseobacterium but can be differentiated from the closely related type strain, C. lathyri RBA2-6 T . The distinctiveness of strain 25-1 T is sufficient to categorize the isolate as a member of a species that is distinguished from the published Chryseobacterium species. The low 16S rRNA gene sequence similarity with all other described Chryseobacterium species supports the description of strain 25-1 T as a member of a new Chryseobacterium species for which we propose the name C. chengduensis sp. nov. ( cheng.du. en’sis . N.L. fem. adj. chengduensis pertaining to Chengdu in Sichuan Province, China, where the type strain was isolated). Cells of strain 25-1 T are non-motile, strictly aerobic, Gram-stain-negative, and rod-shaped (0.44–0.48 μm wide and 0.88–0.92 μm long). Colonies on LB agar are non-transparent, yellow-pigmented, circular, and smooth with regular edges after 3 d of incubation at 30 °C. The non-fluorescent and non-diffusible yellow pigments belong to the flexirubin type. Cells grow in LN medium with 0%–2% NaCl with an optimum concentration of 0%–0.5%. Optimal growth occurs at 28–30 °C and no growth occurs below 10 °C or above 40 °C. Growth occurs at pH 6.0–9.0 and optimally at pH 7.0–8.0. Catalase and oxidase are produced, but not indole or H 2 S. Starch and Tween 80 are degraded but not gelatin. Nitrate is not reduced to nitrite. Acid is not produced from salicin, acetamide, mannose, (-galactosidase, trehalose, cellobiose, arabinose, and lactose. It is negative for adipic acid and arginine decarboxylase, and positive for the production of ornithine decarboxylase. In the Phoenix system, it is positive for arginine-arginine-AMC, glycine-proline-AMC, glycine-AMC, glutaryl-glycine-arginine-AMC, L -arginine-AMC, L -leucine-AMC, L -phenylalanine-AMC, L -pyroglutamic acid-AMC, L -tryptophan-AMC, lysine-alanine-AMC, acetate, adonitol, citrate, colistin, D -mannitol, α-ketoglutaric acid, malonate, tiglic acid, polymyxin B, and 4MU- N -acetyl-(- D -glucosaminide. It is sensitive to vancomycin, tetracycline, rifampicin, ciprofloxacin, florfenicol, doxycycline, amikacin, enrofloxacin, cefotaxime, ceftazidime, furazolidone, and trimesulf, but resistant to kanamycin, spectinomycin, erythromycin, cephalothin, sulfamethoxazole, lincomycin, streptomycin, ampicillin, penicillin, and gentamicin. The major cellular fatty acids are iso -C 15:0 , iso -C 17:0 3OH, summed feature 3 (C 16:1 (7c and/or C 16:1 (6c) and summed feature 9 (10-methyl C 16:0 and/or iso -C 17:1 (9c). The only isoprenoid quinone detected was MK-6. The DNA G+C molar content was 36.2%. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500214 Project supported by the Chengdu Giant Panda Breeding Research Foundation (No. CPF2010-06) and the National Key Technology R & D Program of China (No. 2012BAC01B06) 2015-09-05 2016-07-13 2016-02-25 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 17