Background: Cholesterol is metabolized into various sterol hormones in living organisms. Excessive use of cholesterol results in high amounts of sterol hormones with adverse effects on environment. Objective: The aim of this study was to identify and isolate a bacteria strain for cholesterol degradation. Methods: Bacterial enrichment, cultivation, serial dilution and agar plating were used for bacterium isolation. Isolated bacteria were identified and characterized through morphological, molecular, physiological and biochemical methods. Further, multi substrate metabolism and drug sensitivity tests were carried out on isolated bacteria. Results: Sanger sequencing and BLAST analysis showed high homology (99.78%) of the isolated bacterial strain with Exiguobacterium profundum strain 10C. The isolated bacterial strain was, therefore named EP01. Further analysis showed the strain was rod or spherical shaped, gram negative and peritrichous. In addition, the strain occurred in single or in pairs assembling bacterium, and produced orange or yellow clone on LB agar plate. The strain required a temperature range of 27-45℃ (optimum temperature was 37℃), pH range of 5-11 (optimum pH was 9), and NaCl range of 0% - 5% (optimal concentration of sodium chloride was 1%) for growth. The doubling time of the EP01 under the optimal conditions was 1.22h. EP01 showed positive catalase, amylase and alkaline protease activities. Further, EP01 tested positive for kinetics, hydrogen sulfide, gelatin hydrolysis and mannitol tests. On the contrary, tested negative for oxidase, urease, citrate, Tween-20, nitrate reductase and phenylalanine decarboxylase activities. EP01 metabolized glucose, xylose, raffinose, fructose and lactose. However, EP01 showed no growth on cholesterol, estradiol, testosterone, ergosterol, carbazole, phenanthrene, tetrachlorobiphenol A, 4-hydroxyphenylacetic acid, n-octanoic acid, n-nonanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid and arabinose as the only carbon source. Furthermore, EP01 was sensitive to 1μg/μL penicillin potassium, chloramphenicol and erythromycin, but not to metronidazole. Conclusion: EP01 is a Exiguobacterium profundum strain with strong alkaline resistance. EP01 can be used for environmental remediation to degrade contaminants.

Heqiang Yuan, Hangjie Fu, Chengru Wu, Yuxiao Li, Guorong Yang, Bin DING. Isolation and Identification of Exiguobacterium Profundum Strain from Cholesterol-Enriched Fetal Sample. Frontiers in Medical Science Research (2020) Vol. 2 Issue 4: 71-82. https://doi.org/10.25236/FMSR.2020.020410.

[1] Bhatti HN, Khera RA. Biological transformations of steroidal compounds: a review. Steroids. 2012, 77(12):1267-1290. doi: 10.1016/j.steroids.2012.07.018.

[2] Björkhem I, Eggertsen G. Genes involved in initial steps of bile acid synthesis. Curr Opin Lipidol. 2001, 12(2):97-103. doi: 10.1097/00041433-200104000-00002.

[3] Yang X, Lin H, Zhang Y, He Z, Dai X, Zhang Z, Li Y. Sorption and desorption of seven steroidal synthetic progestins in five agricultural soil-water systems. Ecotoxicol Environ Saf. 2020, 196:110586. doi: 10.1016/j.ecoenv.2020.110586.

[4] Gold DA, Caron A, Fournier GP, Summons RE. Paleoproterozoic sterol biosynthesis and the rise of oxygen. Nature. 2017, 543(7645):420-423. doi: 10.1038/nature21412.

[5] Nie ZQ. Accumulation of steroid hormones in soil and its adjacent aquatic environment from a typical intensive vegetable cultivation of North China. Sci Total Environ. 2015, 538(538):423-430. doi: 10.1016/j.scitotenv.2015.08.067.

[6] Veiga P, Juste C, Lepercq P, Saunier K, Béguet F, Gérard P. Correlation between faecal microbial community structure and cholesterol-to-coprostanol conversion in the human gut. FEMS Microbiol Lett. 2005, 242(1):81-86. doi: 10.1016/j.femsle.2004.10.042.

[7] Frena M, Santos APS, Souza MRR, Carvalho SS, Madureira LAS, Alexandre MR. Sterol biomarkers and fecal coliforms in a tropical estuary: Seasonal distribution and sources. Mar Pollut Bull. 2019, 139:111-116. doi: 10.1016/j.marpolbul.2018.12.007.

[8] Wei ST, Wu YW, Lee TH, Huang YS, Yang CY, Chen YL, Chiang YR. Microbial Functional Responses to Cholesterol Catabolism in Denitrifying Sludge. mSystems. 2018, 3(5): e00113-18. doi: 10.1128/mSystems.00113-18.

[9] Holert J, Cardenas E, Bergstrand LH, Zaikova E, Hahn AS, Hallam SJ, Mohn WW. Metagenomes Reveal Global Distribution of Bacterial Steroid Catabolism in Natural, Engineered, and Host Environments. mBio. 2018, 9(1); e02345-17. doi: 10.1128/mBio.02345-17.

[10] Hudson SB, Kluever BM, Webb AC, French SS. Steroid hormones, energetic state, and immunocompetence vary across reproductive contexts in a parthenogenetic lizard. Gen Comp Endocrinol. 2020, 288:113372. doi: 10.1016/j.ygcen.2019.113372.

[11] Hoffman M, Mann LK, Won JH, Bergh EP, Donepudi R, Johnson A, Moise KJ, Macpherson C, Thom E, Mesiano S, Papanna R. Steroid Hormone Levels in Recipient Amniotic Fluid in Twin-Twin Transfusion Syndrome and Their Association with Preterm Delivery. Am J Perinatol. 2019, 37(6):562-569. doi: 10.1055/s-0039-1700862.

[12] Houshdaran S, Oke AB, Fung JC, Vo KC, Nezhat C, Giudice LC. Steroid hormones regulate genome-wide epigenetic programming and gene transcription in human endometrial cells with marked aberrancies in endometriosis. PLoS Genet. 2020, 16(6): e1008601. doi: 10.1371/journal.pgen.1008601.

[13] Xu P, Zhou X, Xu D, Xiang Y, Ling W, Chen M. Contamination and Risk Assessment of Estrogens in Livestock Manure: A Case Study in Jiangsu Province, China. Int J Environ Res Public Health. 2018, 15(1):125. doi: 10.3390/ijerph15010125.

[14] Qian CR, Huang YX. Laboratory Experiments Microbiology[M]. Beijing: Peking University Press, 1999: 113 (in Chinese)

[15] Kim IG, Lee MH, Jung SY, Song JJ, Oh TK, Yoon JH. Exiguobacterium aestuarii sp. nov. and Exiguobacterium marinum sp. nov., isolated from a tidal flat of the Yellow Sea in Korea [J]. Int J Sytm Evol Microbiol, 2005,55: 885-889.doi: 10.1099/ijs.0.63308-0

[16] Zhu Daochen, Xie Changxiao, Sun Jianzhong, et.al. A deep exiguobacterium profundum and its application in degradation of organic pollutants, China. CN201510010845.3 .2018.02.27

[17] Miteva VI, Sheridan PP, Brenchly JE. Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier icecore [J]. Appl Environ Microbiol, 2004, 70: 5337-5363. DOI:10.1128/AEM.70.1.202-213.2004

[18] Rodriguez DF, Goris J, Vishnivetskaya T, Gilichinsky D, Thomashow MF, Tiedje JM. Characterization of Exiguobacterium isolates from the Siberian permafrost. Description of Exiguobacterium sibiricum sp. Nov [J]. Extrenophiles, 2006, 10: 285-294. DOI:10.1007/s00792-005-0497-5

[19] SelvakumarC, Joshi P, Nazim S, Mishra PK, Kundu S, Gupta HS. Exiguobacterium acetylicum strain 1P (MTCC 8707) a novel bacterial antagonist from the North Western Indian Himalayas [J]. World J Microbiol Biotechnol, 2009, 25: 131-137. DOI:10.1007/s11274-008-9874-4

[20] Xia Xiaomin, Wang Jianjun, Chen Liqi, Zhang Rui. Preliminary study on bacterial community structure in atmospheric aerosols in Xiamen City in October [J]. Journal of Xiamen University (Natural Science), 2010, 49 (5): 682-687. DOI:CNKI: SUN: XDZK.0.2010-05-020

[21] Collins MD, Lund BM, Farrow JAE, et al. Chemotaxonomic study of an alkalophilic bacterium, Exiguobacterium aurantiacum gen nov., sp. nov. J Gen Microbiol. 1983; 129:2037–2042. DOI:10.1099/00221287-129-7-2037

[22] Davidson MW, Gray KA, Knaff DB, Krulwich TA. Purification and characterization of two soluble cytochromes from the alkalophile Bacillus firmus RAB. Biochim Biophys Acta. 1988 May 11;933(3):470-7. doi: 10.1016/0005-2728(88)90082-5.

[23] Chaturvedi P, Prabahar V, Manorama R, Pindi PK, Bhadra B, Begum Z,Shivaji S. Exiguobacterium soli sp. nov., a psychrophilic bacterium from the McMurdo Dry Valleys, Antarctica [J]. Int J Sytm Evol Microbiol, 2008, 58: 2447-2453. DOI:10.1099/ijs.0.2008/000067-0.

[24] Fruhling A, Schumann P, Hippe H, Straubler B, Stackebrandt E. Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp.nov [J]. Int J Sytm Evol Microbiol, 2002, 52: 1171-1176. DOI:10.1007/s10482-009-9356-1

[25] Crapart S, Fardeau ML, Cayol JL, Thomas P, Sery C, Ollivier B, Combet-Blanc Y. Exiguobacterium profundum sp. nov., a moderately thermophilic, lactic acid-producing bacterium isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol. 2007, 57(2):287-292. doi: 10.1099/ijs.0.64639-0. DOI:10.1099/ijs.0.64639-0

[26] Anbu P, Hur BK, Gyun Lee C. Isolation and characterization of a novel oxidant- and surfactant-stable extracellular alkaline protease from Exiguobacterium profundum BK-P23. Biotechnol Appl Biochem. 2013, 60(2):155-161. doi: 10.1002/bab.1059.

[27] Zhang Ying, Shi Ping, MA Jiong. Research Progress on Exiguobacterium spp. and its environmental application [J]. Journal of Applied and Environmental Biology, 2013(5):898-904. DOI:10.3724/sp.j.1145.2013.00898