Shewanella Chilikensis Research Articles

Shewanella chilikensis MG22 isolated from tannery site for malachite green decolorization in microbial fuel cell: a proposed solution for recirculating aquaculture system (RAS)

Malachite Green (MG) dye of the triphenylmethane group is a toxic compound used in the aquaculture industry as an antifungal agent, however, it can accumulate in fish and pose toxicity. The present work aims to remove MG in Microbial Fuel Cell (MFC) as a sustainable and eco-friendly solution. Out of six samples, the highest malachite green degradation was obtained by a sample obtained from Robiki tannery site in agar plates in 24 h at 37 °C. Robiki sample was used to inoculate the anodic chamber in Microbial Fuel cell, the resulting average electricity production was 195.76 mV for two weeks. The decolorization average was almost 88%. The predominant bacteria responsible for MG decolorization and electricity production were identified using 16S rRNA as Shewanella chilikensis strain MG22 (Accession no. OP795826) and formed a heavy biofilm on the anode. At the end of the decolorization process, MG was added again for re-use of water. The results showed efficiency for re-use 3 times. To ensure the sterility of treated water for re-use, both UV and filter sterilization were used, the latter proved more efficient. The obtained results are promising, MFC can be used as recirculating aquaculture system (RAS). The same aquaculture water can be treated multiple times which provides a sustainable solution for water conservation.Graphical

Integration of microbiome and Koch’s postulates to reveal multiple bacterial pathogens of whitish muscle syndrome in mud crab, Scylla paramamosain

BackgroundFor more than a century, the Koch’s postulates have been the golden rule for determining the causative agents in diseases. However, in cases of multiple pathogens-one disease, in which different pathogens can cause the same disease, the selection of microorganisms that regress infection is hard when Koch’s postulates are applied. Microbiome approaches can obtain relatively complete information about disease-related microorganisms and can guide the selection of target microorganisms for regression infection. In the present study, whitish muscle syndrome (WMS) of Scylla paramamosain, which has typical symptoms with whitish muscle and blackened hemolymph was used as an example to establish a new research strategy that integrates microbiome approaches and Koch’s postulates to determinate causative agents of multiple pathogens-one disease.ResultsMicrobiome results revealed that Aeromonas, Acinetobacter, Shewanella, Chryseomicrobium, Exiguobacterium, Vibrio and Flavobacterium, and Kurtzmaniella in hemolymph were bacterial and fungal indicators for WMS. A total of 23 bacteria and 14 fungi were isolated from hemolymph and muscle tissues, and among the bacteria, Shewanella chilikensis, S. xiamenensis, Vibrio alginolyticus, S. putrefaciens, V. fluvialis, and V. parahaemolyticus were present in hemolymph and/or muscle tissues in each WMS crab, and the last three species were also present in three Healthy crabs. The target bacteria and fungi were further screened to regression infections based on two criteria: whether they belonged to the indicator genera for WMS, whether they were isolated from both hemolymph and muscle tissues in most WMS crabs. Only S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria. The six bacteria that met both two criteria and six fungi and another bacterium that unmatched any of two criteria were used to perform regression infection experiments based on Koch’s postulates. S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria, and the results indicate that they cause WMS in crabs independently.ConclusionsThis study fully demonstrated that our research strategy that integrates the microbiome and Koch’s postulates can maximize the ability to catch pathogens in one net for the situation of multiple pathogens-one disease.EgKJnBsvf5xgzELdpRDt_QVideo

Corrosion of Carbon Steel by Shewanella chilikensis DC57 Under Thiosulphate and Nitrate Reducing Conditions.

Shewanella chilikensis DC57 is a bacterial strain isolated from a corrosion failure in a floating oil production system. Previous studies have indicated that this microorganism has potential to trigger corrosion of carbon steel through several metabolic pathways identified in its genome. In this study we evaluated the corrosion of carbon steel by S. chilikensis in the presence of thiosulphate or nitrate as terminal electron acceptors of the anaerobic respiration. Electrochemical response of carbon steel to the biofilm formation revealed differences in the corrosion process under the different electron acceptors conditions. Microscopic examination of the metal surface confirmed that S. chilikensis induced corrosion in both scenarios; however, in the presence of thiosulfate S. chilikensis triggered a higher pitting corrosion rate, whereas in presence of nitrate it promoted higher uniform corrosion. This study demonstrates the importance of understanding the metabolic versatility of microbes in order to assess the MIC risk of industrial facilities.

QnrA gene diversity in Shewanella spp.

Members of Shewanella are ubiquitous in aquatic environments, some of which have been implicated in human infections. The progenitors of antibiotic resistance genes with clinical relevance, such as qnrA genes, have been identified in Shewanella. qnrA code for a pentapeptide repeat protein that protects type II topoisomerases, decreasing susceptibility to quinolones and fluoroquinolones. In this study, 248 genomes of 49 Shewanella species were analysed as well as 33 environmental isolates belonging to 10 Shewanella species. The presence of the qnrA gene was detected in 22.9% of the genomes and 15.2% of the isolates. The gene was more often detected in Shewanella algae, but was also detected in Shewanella carassii, Shewanella chilikensis, Shewanella haliotis and Shewanella indica. The identified genes encoded the previously described variants QnrA3 (in 22 genomes of one species), QnrA2 (eight genomes and three species), QnrA1 (six genomes and two species), QnrA7 (five genomes and two species), QnrA10 (two genomes of one species) and QnrA4 (one genome). In addition, 11 novel variants with 3 to 7 amino acid substitutions were identified (in 13 genomes and one environmental isolate). The presence of this gene appears to be species-specific although within some species several variants were detected. The study presents a previously unknown diversity of qnrA in Shewanella, highlighting the role of this genus as progenitor and reservoir of these genes. Further studies are needed to determine the phenotypes conferred by the new variants and the mechanisms that may mediate the transfer of these genes to new hosts.

Genetic Environment Surrounding blaOXA-55-like in Clinical Isolates of Shewanella algae Clade and Enhanced Expression of blaOXA-55-like in a Carbapenem-Resistant Isolate.

ABSTRACTAlthough Shewanella spp. are most frequently isolated from marine environments; more rarely, they have been implicated in human infections. Shewanella spp. are also recognized as the origin of genes for carbapenem-hydrolyzing class D β-lactamases. Due to the spread globally among Enterobacterales in recent years, risk assessments of both clinical and environmental Shewanella strains are urgently needed. In this study, we analyzed the whole-genome sequences of 10 clinical isolates and 13 environmental isolates of Shewanella spp. and compared them with those of Shewanella species strains registered in public databases. In addition, the levels of blaOXA-55-like transcription and β-lactamase activity of a carbapenem-resistant Shewanella algae isolate were compared with those of carbapenem-susceptible S. algae clade isolates. All clinical isolates were genetically identified as S. algae clade (S. algae, Shewanella chilikensis, and Shewanella carassii), whereas all but one of the environmental isolates were identified as various Shewanella spp. outside the S. algae clade. Although all isolates of the S. algae clade commonly possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, genetic structures outside this region were different among species. Among S. algae clade isolates, only one showed carbapenem resistance, and this isolate showed a high level of blaOXA-55-like transcription and β-lactamase activity. Although this study documented the importance of the S. algae clade in human infections and the relationship between enhanced production of OXA-55-like and resistance to carbapenems in S. algae, further studies are needed to elucidate the generalizability of these findings.IMPORTANCE Shewanella spp., which are known to carry chromosomally located blaOXA genes, have mainly been isolated from marine environments; however, they can also cause infections in humans. In this study, we compared the molecular characteristics of clinical isolates of Shewanella spp. with those originating from environmental sources. All 10 clinical isolates were genetically identified as members of the Shewanella algae clade (S. algae, S. chilikensis, and S. carassii); however, all but one of the 13 environmental isolates were identified as Shewanella species members outside the S. algae clade. Although all the S. algae clade isolates possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, only one isolate showed carbapenem resistance. The carbapenem-resistant isolate showed a high level of blaOXA-55-like transcription and β-lactamase activity compared with the carbapenem-susceptible isolates. To confirm the clinical significance and antimicrobial resistance mechanisms of the S. algae clade members, analysis involving more clinical isolates should be performed in the future.

Evaluation of a novel, multi-functional inhibitor compound for prevention of biofilm formation on carbon steel in marine environments

Chemical biocides remain the most effective mitigation strategy against microbiologically influenced corrosion (MIC), one of the costliest and most pervasive forms of corrosion in industry. However, toxicity and environmental concerns associated with these compounds are encouraging the development of more environmentally friendly MIC inhibitors. In this study, we evaluated the antimicrobial effect of a novel, multi-functional organic corrosion inhibitor (OCI) compound, cetrimonium trans-4-hydroxy-cinnamate (CTA-4OHcinn). Attachment of three bacterial strains, Shewanella chilikensis, Pseudomonas balearica and Klebsiella pneumoniae was evaluated on wet-ground (120 grit finish) and pre-oxidised carbon steel surfaces (AISI 1030), in the presence and absence of the new OCI compound. Our study revealed that all strains preferentially attached to pre-oxidised surfaces as indicated by confocal laser scanning microscopy, scanning electron microscopy and standard colony forming unit (CFU) quantification assays. The inhibitor compound at 10 mM demonstrated 100% reduction in S. chilikensis attachment independent of initial surface condition, while the other two strains were reduced by at least 99.7% of the original viable cell number. Our results demonstrate that CTA-4OHcinn is biocidal active and has promise as a multifunctional, environmentally sound MIC inhibitor for industrial applications.

Establishment and Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Detection of Shewanella Genus.

Shewanella species are widely distributed in the aquatic environment and aquatic organisms. They are opportunistic human pathogens with increasing clinical infections reported in recent years. However, there is a lack of a rapid and accurate method to identify Shewanella species. We evaluated here matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of Shewanella. A peptide mass reference spectra (PMRS) database was constructed for the type strains of 36 Shewanella species. The main spectrum projection (MSP) cluster dendrogram showed that the type strains of Shewanella species can be effectively distinguished according to the different MS fingerprinting. The PMRS database was validated using 125 Shewanella test strains isolated from various sources and periods; 92.8% (n = 116) of the strains were correctly identified at the species level, compared with the results of multilocus sequence analysis (MLSA), which was previously shown to be a method for identifying Shewanella at the species level. The misidentified strains (n = 9) by MALDI-TOF MS involved five species of two groups, i.e., Shewanella algae–Shewanella chilikensis–Shewanella indica and Shewanella seohaensis–Shewanella xiamenensis. We then identified and defined species-specific biomarker peaks of the 36 species using the type strains and validated these selected biomarkers using 125 test strains. Our study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid identification of Shewanella strains at the species level.

Complete Genome Sequence of Shewanella chilikensis Strain DC57, Isolated from Corroded Seal Rings at a Floating Oil Production System in Australia.

Here, we describe the genome of Shewanella chilikensis strain DC57, a facultatively anaerobic bacterium isolated from corroded seal rings at a floating oil production system in Australia. The genome of strain DC57 has a size of 4.91 Mbp and harbors 4,178 predicted protein-encoding genes.

Carbon steel corrosion by bacteria from failed seal rings at an offshore facility

Corrosion of carbon steel by microorganisms recovered from corroded seal rings at an offshore floating production facility was investigated. Microbial diversity profiling revealed that communities in all sampled seal rings were dominated by Pseudomonas genus. Nine bacterial species, Pseudomonas aeruginosa CCC-IOB1, Pseudomonas balearica CCC-IOB3, Pseudomonas stutzeri CCC-IOB10, Citrobacter youngae CCC-IOB9, Petrotoga mobilis CCC-SPP15, Enterobacter roggenkampii CCC-SPP14, Enterobacter cloacae CCC-APB1, Cronobacter sakazakii CCC-APB3, and Shewanella chilikensis CCC-APB5 were isolated from corrosion products and identified based on 16S rRNA gene sequence. Corrosion rates induced by the individual isolates were evaluated in artificial seawater using short term immersion experiments at 40 °C under anaerobic conditions. P. balearica, E. roggenkampii, and S. chilikensis, which have not been associated with microbiologically influenced corrosion before, were further investigated at longer exposure times to better understand their effects on corrosion of carbon steel, using a combination of microbiological and surface analysis techniques. The results demonstrated that all bacterial isolates triggered general and localised corrosion of carbon steel. Differences observed in the surface deterioration pattern by the different bacterial isolates indicated variations in the corrosion reactions and mechanisms promoted by each isolate.

Isolation, Screening and Characterization of Exopolysaccharide Producing Bacteria

Exopolysaccharides(EPSs) are essential metabolites synthesized and excreted by certain microorganisms in response to extreme condition of pH, temperature, salinity, osmotic stress and other contaminants for survival in such adverse environment. The present study focuses on isolation of exopolysaccharide producing bacteria from extreme environment of oil polluted soil of Ogoni land and marine water of Bonny Island. Screening of EPS producing abilities of the selected isolates were estimated using two approaches viz gravimetric analysis of EPS dry weight and quantification assay for total carbohydrate content by phenol sulphuric acid method. Result revealed that a total of forty (40) different colonies were suspected to produce exopolysaccharide after preliminary screening by selecting thick ropy-like colony formers on agar medium. Secondary screening indicated that twelve (12) isolates produced precipitates above 1000mg/l of total dry weight and eight (8) isolates produced highest EPS yield above 1000mg/l of culture media with carbohydrate content determination. Five isolates with the code WAS1, WAS11, SC6, SOS7 and SOS10 produced significantly higher EPS compared to other bacterial colonies isolated and were termed as most potent EPS producers. These isolates were identified based on 16S rDNA sequence as Providencia stuarti, Escherichia coli, Shewanella chilikensis and Bacillus nealsonii. The result of the present study indicated that these strains have the potentials of producing high exopolysaccharides and can be explored in biotechnological industries.

Identification and optimization of biosurfactant producing bacteria isolated from rag layer crude oil emulsion

Biosurfactants are surface active compounds that synthesized as secondary metabolite by wide range of bacteria and have characteristic in lowering surface and interfacial tension. This study aimed to isolate and identify biosurfactant producing bacteria from rag layer crude oil emulsion. Rag layer is considered as undesirable material as it is difficult to be separated because of the stable interaction between different components. Mineral salt medium supplemented with glucose and crude oil was used to screen the ability of isolates to produce biosurfactant. Five bacterial strains that successfully isolated from rag layer crude oil emulsion sample were screened for hydrocarbon degradation and biosurfactant production. Two isolates shown positive results in drop collapse test, surface tension measurement and emulsification index, namely P3b and P4. 16S rRNA analysis revealed P3b and P4 to be closely related to Enterobacter xiangfangensis while P4 was Shewanella chilikensis, respectively. Only isolate P3b was selected for further study. Enterobacter xiangfangensis SSP3b16 was found to grow optimally at 37°C, pH 7.0 and 10mM glucose. The highest reduction of surface tension was recorded when culture medium supplemented with 7 % (v/v) glucose and 2 g/L ammonium nitrate. From this study, the biosurfactant production by Enterobacter xiangfangensis SSP3b16 can potentially be exploited to enhance oil recovery as well as in solving the rag layer problem in oil industries.

Shewanella carassii sp. nov., isolated from surface swabs of crucian carp and faeces of a diarrhoea patient.

Two strains, 08MAS2251T and LZ2016-166, were isolated from diverse samples in China collected from the surface of crucian carp and the faeces of a diarrhoea patient, respectively. Both strains were pink-orange coloured, Gram-negative, oxidase- and catalase-positive, facultative anaerobic and motile bacteria, produced H2S and reduced nitrates to nitrites. Growth occurred in the presence of 0-9 % (w/v) NaCl and at 10-42 °C. The optimum conditions were with 1 % (w/v) NaCl and at 35 °C. The phylogenetic tree of 16S rRNA gene demonstrated that strains 08MAS2251T and LZ2016-166 clustered in a distinctive clade next to the species Shewanella chilikensis JC5T within the genus Shewanella. Meanwhile, gyrB gene sequence analysis indicated that the two strains formed an independent branch that was clearly separate from all the other Shewanella species with sequence similarities from 68.49 to 95.74 %. The DNA G+C content of strain 08MAS2251T was 52.68 mol%. Genomic relatedness of in silico DNA-DNA hybridization between strain 08MAS2251T and phylogenetic neighbours ranged from 50.5-51.8 %, below the cutoff of 70 %. In addition, corresponding average nucleotide identity values were between 93.01 to 93.49%, which were lower than 95 % threshold. The major fatty acids of strain 08MAS2251T were C17 : 1ω8c (27.2 %), iso-C15 : 0 (22.5 %), summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 8.7 %), C16 : 0 (6.2 %), iso-C13 : 0 (5.6 %) and C17 : 0 (4.5 %). Based on phenotypic and genetic analysis, strains 08MAS2251T and LZ2016-166 are identified as a novel species of the genus Shewanella, for which the name Shewanellacarassii sp. nov. is proposed. The type strain is 08MAS2251T (=DSM 104682T=CGMCC 1.16033T).

Microbial diversity of saline environments: searching for cytotoxic activities

In order to select halophilic microorganisms as a source of compounds with cytotoxic activities, a total of 135 bacterial strains were isolated from water and sediment samples collected from the Zipaquirá salt mine in the Colombian Andes. We determined the cytotoxic effects of 100 crude extracts from 54 selected organisms on the adherent murine mammary cell carcinoma 4T1 and human mammary adenocarcinoma MCF-7 cell lines. These extracts were obtained from strains of Isoptericola, Ornithinimicrobium, Janibacter, Nesterenkonia, Alkalibacterium, Bacillus, Halomonas, Chromohalobacter, Shewanella, Salipiger, Martellela, Oceanibaculum, Caenispirillum and Labrenzia. The extracts of 23 strains showed an IC50 of less than 100 μg mL−1. They were subsequently analyzed by LC/MS allowing dereplication of 20 compounds. The cytotoxic effect was related to a complex mixture of diketopiperazines present in many of the extracts analyzed. The greatest cytotoxic activity against both of the evaluated cell lines was obtained from the chloroform extract of Labrenzia aggregata USBA 371 which had an IC50 < 6 μg mL−1. Other extracts with high levels of cytotoxic activity were obtained from Bacillus sp. (IC50 < 50 μg mL−1) which contained several compounds such as macrolactin L and A, 7-O-succinoylmacrolactin F and iturin. Shewanella chilikensis USBA 344 also showed high levels of cytotoxic activity against both cell lines in the crude extract: an IC50 < 15 μg mL−1 against the 4T1 cell line and an IC50 < 68 μg mL−1 against the MCF-7 cell line. Nesterenkonia sandarakina CG 35, which has an IC50 of 118 µg mL−1 against 4T1, is a producer of diketopiperazines and 1-acetyl-β-carboline. Also, Ornithinimicrobium kibberense CG 24, which has IC50 < 50 μg mL−1, was a producer of diketopiperazines and lagunamycin. Our study demonstrates that these saline environments are habitats of halophilic and halotolerant bacteria that have previously unreported cytotoxic activity.

Shewanella upenei sp. nov., a lipolytic bacterium isolated from bensasi goatfish Upeneus bensasi

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R(T), was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R(T) belonged to the genus Shewanella. Strain 20-23R(T) exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192(T), Shewanella haliotis DW01(T), and Shewanella chilikensis JC5(T), respectively. Strain 20-23R(T) exhibited 93.1-96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3-98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R(T) contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20-23R(T), S. algae KCTC 22552(T) and S. haliotis KCTC 12896(T) were similar; major components were iso-C(15:0), C(16:0), C(16:1) ω7c and/or iso-C(15:0) 2-OH and C(17:1) ω8c. The DNA G+C content of strain 20-23R(T) was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20-23R(T), together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R(T) represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20-23R(T) (=KCTC 22806(T) =CCUG 58400(T)).

Shewanella indica sp. nov., isolated from sediment of the Arabian Sea

A Gram-negative, facultatively anaerobic, rod-shaped, catalase- and oxidase-positive bacterium, motile by means of a single polar flagellum and designated strain KJW27(T), was isolated from the marine sediment of Karwar jetty, west coast of India. The strain was β-haemolytic and grew with 0-10 % (w/v) NaCl, at 10-45 °C and at pH 6.5-10, with optimum growth with 2 % (w/v) NaCl, at 37 °C and at pH 7.5. The major fatty acids were iso-C₁₅:₀ (22.2 %), C₁₇:₁ω8c (21 %), summed feature 3 (comprising C₁₆:₁ω7c and/or C₁₆:₁ω6c; 10.2 %), C₁₆:₀ (7.1 %), iso-C₁₃:₀ (5.6 %) and C₁₇:₀ (4.4 %). The DNA G+C content was 51.2 mol%. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences showed that strain KJW27(T) forms a lineage within the genus Shewanella and is closely related to Shewanella algae ATCC 51192(T) (98.8 %), Shewanella haliotis DW01(T) (98.8 %) and Shewanella chilikensis JC5(T) (98.2 %). Sequence identity with other members of this genus ranges from 92.2 to 96.4 %. The DNA-DNA relatedness of strain KJW27(T) with S. algae ATCC 51192(T), S. haliotis DW01(T) and S. chilikensis JC5(T) was 52, 44 and 33 %, respectively. The phenotypic, genotypic and DNA-DNA relatedness data indicate that strain KJW27(T) should be distinguished from S. algae ATCC 51192(T), S. haliotis DW01(T) and S. chilikensis JC5(T). On the basis of the data presented in this study, strain KJW27(T) represents a novel species, for which the name Shewanella indica sp. nov. is proposed. The type strain is KJW27(T) ( = KCTC 23171(T) = BCC 41031(T) = NCIM 5388(T)).

Shewanella chilikensis sp. nov., a moderately alkaliphilic gammaproteobacterium isolated from a lagoon

A Gram-negative, motile, rod-shaped, facultatively anaerobic bacterium was isolated from sediment of Chilika Lagoon, a brackish water lagoon in India. The strain, designated JC5(T), was able to grow in the presence of 0-8.0 % NaCl and at pH 7.0-10.0. The isolate was positive for oxidase and catalase and exhibited alpha-haemolysis. The major fatty acids were iso-C(15 : 0) (18.3 %), C(16 : 0) (11.3 %), C(17 : 1)omega8c (13.1 %) and a summed feature, C(16 : 1)omega7c and/or C(16 : 1)omega6c (15.1 %). The genomic DNA G+C content was 54.6 mol%. A phylogenetic tree based on the 16S rRNA gene sequences showed that strain JC5(T) forms a lineage within the genus Shewanella and is closely related to Shewanella haliotis DW01(T) (98.0 %), Shewanella algae ATCC 51192(T) (97.6 %) and Shewanella marina C4(T) (95.8 %). Further, genomic DNA-DNA hybridization of strain JC5(T) with S. haliotis DW01(T) and S. algae ATCC 51192(T) showed relatedness of only 42 and 23 %, respectively. On the basis of phenotypic and molecular genetic evidence, strain JC5(T) represents a novel species of the genus Shewanella, for which the name Shewanella chilikensis sp. nov. is proposed. The type strain is JC5(T) (=CCUG 57101(T) =NBRC 105217(T) =KCTC 22540(T)).