微生物菌群培养组学在动物医学中的应用

doi:10.11843/j.issn.0366-6964.2020.12.004

Abstract

Abstract: Under laboratory conditions, the number of cultured microorganisms accounts for about 1% of the total number of microorganisms in nature, which limits people's understanding and utilization of 99% of the unknown microorganisms. However, relevant researches show that those "uncultured microorganisms" can be developed and utilized, and the uncultured microorganisms are the main body of the unknown microorganisms. The microbial culturomics explored the application of multiple culture conditions and long-term culture, it was combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and 16S ribosomal RNA (rRNA) sequencing to identify all kinds of microorganisms on a large scale. At the same time, whole-genome sequencing (WGS) and Metagenomics sequencing technology were used to analyze unknown microorganisms in depth. In this paper, the latest progress of culturomics in the ruminant gastrointestinal tract, poultry cecum, and livestock nasal microflora in recent years was reviewed, and the feasibility of applying the method of microflora culturomics in animal disease prevention and control was discussed. As a new research idea, culturomics has some immature aspects, but its development prospect is very broad. The complementary of microflora culturomics and other research methods have gradually become a breakthrough in the development of veterinary microbiology.

Key words: culturomics, MALDI-TOF-MS, 16S rRNA, genome sequencing, rumen microflora, cecal gut microflora, nasal microbiota

CLC Number:

S852.6

PENG Na, PENG Xianqi, YUE Min. The Application of Microbiome Culturomics in Veterinary Medicine[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 2942-2953.

References 70

[1]	TORSVIK V, ØVREÅS L. Microbial diversity and function in soil:from genes to ecosystems[J]. Curr Opin Microbiol, 2002, 5(3):240-245.
[2]	何彪,涂长春.病毒宏基因组学的研究现状及应用[J].畜牧兽医学报,2012,43(12):1865-1870.HE B, TU C C.The advances and applications of viral metagenomics[J].Acta Veterinaria et Zootechnica Sinica, 2012,43(12):1865-1870.(in Chinese)
[3]	TRAORÉ S, BILEN M, CADORET F, et al. Study of human gastrointestinal microbiota by culturomics in Africa[J]. Med Sante Trop, 2019, 29(4):366-370.
[4]	綦世金, 桂干北, 曾泽, 等. 瘤胃微生物培养组学研究及Hungate1000计划项目进展[J]. 农技服务, 2019, 36(12):36-38.QI S J, GUI G B, ZENG Z, et al. Research on rumen microbiological culture and progress of Hungate1000 project[J]. Agricultural Technology Service, 2019, 36(12):36-38. (in Chinese)
[5]	牛尚博, 蔡嘉裕, 韦金涛, 等. 人体肠道细菌的培养组学研究进展[J]. 生态科学, 2020, 39(2):227-232.NIU S B, CAI J Y, WEI J T, et al. Research progress in human gut bacteria culturomics[J]. Ecological Science, 2020, 39(2):227-232. (in Chinese)
[6]	LAGIER J C, ARMOUGOM F, MILLION M, et al. Microbial culturomics:paradigm shift in the human gut microbiome study[J]. Clin Microbiol Infect, 2012, 18(12):1185-1193.
[7]	GRÉGORY D, CHAUDET H, LAGIER J C, et al. How mass spectrometric approaches applied to bacterial identification have revolutionized the study of human gut microbiota[J]. Expert Rev Proteomics, 2018, 15(3):217-229.
[8]	MARTELLACCI L, QUARANTA G, PATINI R, et al. A literature review of metagenomics and culturomics of the peri-implant microbiome:current evidence and future perspectives[J]. Materials (Basel), 2019, 12(18):3010.
[9]	BILEN M, MBOGNING FOUNKOU M D, CADORET F, et al. Sanguibacter massiliensis sp. nov., Actinomyces minihominis sp. nov., Clostridium minihomine sp. nov., Neobittarella massiliensis gen. nov. and Miniphocibacter massiliensis gen. nov., new bacterial species isolated by culturomics from human stool samples[J]. New Microbes New Infect, 2018, 24:21-25.
[10]	ANGELAKIS E, LAGIER J C. Samples and techniques highlighting the links between obesity and microbiota[J]. Microb Pathog, 2017, 106:119-126.
[11]	LAGIER J C, HUGON P, KHELAIFIA S, et al. The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota[J]. Clin Microbiol Rev, 2015, 28(1):237-264.
[12]	LAGIER J C, EDOUARD S, PAGNIER I, et al. Current and past strategies for bacterial culture in clinical microbiology[J]. Clin Microbiol Rev, 2015, 28(1):208-236.
[13]	ALHANOUT K, MALESINKI S, VIDAL N, et al. New insights into the antibacterial mechanism of action of squalamine[J]. J Antimicrob Chemother, 2010, 65(8):1688-1693.
[14]	CHAPIN K C, LAUDERDALE T. Reagents, stains and media:bacteriology[M]//MURRAY P R, BARON E J, JORGENSEN J H, et al. Manual of Clinical Microbiology[M]. 8th ed. Washington, DC:ASM Press, 2003:354-393.
[15]	LAGIER J C, RAMASAMY D, RIVET R, et al. Non contiguous-finished genome sequence and description of Cellulomonas massiliensis sp. nov.[J]. Stand Genomic Sci, 2012, 7:258-270.
[16]	HECKER M, SCHUMANN W, VOLKER U. Heat-shock and general stress response in Bacillus subtilis[J]. Mol Microbiol, 1996, 19(3):417-428.
[17]	SUBRAMANYAM B, SIVARAMAKRISHNAN G N, DUSTHACKEER A, et al. Phage lysin as a substitute for antibiotics to detect Mycobacterium tuberculosis from sputum samples with the BACTEC MGIT 960 system[J]. Clin Microbiol Infect, 2012, 18(5):497-501.
[18]	GOODMAN A L, KALLSTROM G, FAITH J J, et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice[J]. Proc Natl Acad Sci U S A, 2011, 108(15):6252-6257.
[19]	ELSHAHED M S, YOUSSEF N H, LUO Q W, et al. Phylogenetic and metabolic diversity of Planctomycetes from Anaerobic, sulfide-and sulfur-Rich Zodletone Spring, Oklahoma[J]. Appl Environ Microbiol, 2007, 73(15):4707-4716.
[20]	EDWARD D G, FITZGERALD W A. Cholesterol in the growth of organisms of the pleuropneumonia group[J]. J Gen Microbiol, 1951, 5(3):576-586.
[21]	CHABAN B, HILL J E. A ‘universal’ type Ⅱ chaperonin PCR detection system for the investigation of Archaea in complex microbial communities[J]. ISME J, 2012, 6(2):430-439.
[22]	张华琦. 瘤胃发酵及其调控[J]. 饲料研究, 2008(6):13-15.ZHANG H Q. Rumen fermentation and its regulation[J]. Feed Research, 2008(6):13-15. (in Chinese)
[23]	卢玉飞, 周凌云, 赵圣国, 等. 近10年瘤胃微生物分离培养研究进展[J]. 中国微生态学杂志, 2012, 24(9):856-861.LU Y F, ZHOU L Y, ZHAO S G, et al. Progress on isolation and culture of rumen microorganisms in recent ten years[J]. Chinese Journal of Microecology, 2012, 24(9):856-861. (in Chinese)
[24]	GUAN L L, NKRUMAH J D, BASARAB J A, et al. Linkage of microbial ecology to phenotype:correlation of rumen microbial ecology to cattle's feed efficiency[J]. FEMS Microbiol Lett, 2008, 288(1):85-91.
[25]	FRIEDMAN N, SHRIKE E, GOLD B. Diet-induced changes of redox potential underlie compositional shifts in the rumen archaeal community[J]. Environ Microbiol, 2017, 9(1):174-184.
[26]	ZEHAVI T, PROBST M, MIZRAHI I, et al. Insights into culturomics of the rumen microbiome[J]. Front Microbiol, 2018, 9:1999.
[27]	DRANCOURT M, BOLLET C, CARLIOZ A, et al. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates[J]. J Clin Microbiol, 2000, 38(10):3623-3630.
[28]	卓娜, 伊丽, 浩斯娜, 等. 基于16S rRNA基因序列分析法比较苏尼特双峰驼和阿拉善双峰驼自然发酵酸驼乳的微生物多样性[J]. 微生物学报, 2019, 59(10):1948-1959.ZHUO N, YI L, HAO S N, et al. Application of 16S rRNA high-throughput sequencing for comparative study of the microbial diversity of traditional fermented bactrian camel milk from Alxa bactrian camel and Sonid bactrian camel[J]. Acta Microbiologica Sinica, 2019, 59(10):1948-1959. (in Chinese)
[29]	MAIDAK B L, COLE J R, LILBURN T G, et al. The RDP-Ⅱ (Ribosomal Database Project)[J]. Nucleic Acids Res, 2001, 29(1):173-174.
[30]	SENG P, DRANCOURT M, GOURIET F, et al. Ongoing revolution in bacteriology:routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry[J]. Clin Infect Dis, 2009, 49(4):543-551.
[31]	邢进, 刘娜, 冯育芳, 等. 基质辅助激光解吸电离飞行时间质谱检测实验动物病原菌效果初探[J]. 中国比较医学杂志, 2020, 30(3):83-88, 97.XING J, LIU N, FENG Y F, et al. Evaluation of the biotyper MALDI-TOF MS system for identification of pathogenic bacteria in laboratory animals[J]. Chinese Journal of Comparative Medicine, 2020, 30(3):83-88, 97. (in Chinese)
[32]	MCCORMACK U M, CURIÃO T, BUZOIANU S G, et al. Exploring a possible link between the intestinal microbiota and feed efficiency in pigs[J]. Appl Environ Microbiol, 2017, 83(15):e00380-17.
[33]	MARCHESI J R, ADAMS D H, FAVA F, et al. The gut microbiota and host health:a new clinical frontier[J]. Gut, 2016, 65(2):330-339.
[34]	APAJALAHTI J, KETTUNEN A, GRAHAM H. Characteristics of the gastrointestinal microbial communities, with special reference to the chicken[J]. World's Poult Sci, 2004, 60(2):223-232.
[35]	OAKLEY B B, LILLEHOJ H S, KOGUT M H, et al. The chicken gastrointestinal microbiome[J]. FEMS Microbiol Lett, 2014, 360(2):100-112.
[36]	HUANG P, ZHANG Y, XIAO K P, et al. The chicken gut metagenome and the modulatory effects of plant-derived benzylisoquinoline alkaloids[J]. Microbiome, 2018, 6(1):211.
[37]	BARNES E M, MEAD G C, BARNUML D A, et al. The intestinal flora of the chicken in the period 2 to 6 weeks of age, with particular reference to the anaerobic bacteria[J]. Br Poult Sci, 1972, 13(3):311-326.
[38]	VIDENSKA P, SEDLAR K, LUKAC M, et al. Succession and replacement of bacterial populations in the caecum of egg laying hens over their whole life[J]. PLoS One, 2014, 9(12):e115142.
[39]	STANLEY D, HUGHES R J, MOORE R J. Microbiota of the chicken gastrointestinal tract:influence on health, productivity and disease[J]. Appl Microbiol Biotechnol, 2014, 98(10):4301-4310.
[40]	FERRARIO C, ALESSANDRI G, MANCABELLI L, et al. Untangling the cecal microbiota of feral chickens by culturomic and metagenomic analyses[J]. Environ Microbiol, 2017, 19(11):4771-4783.
[41]	HIERGEIST A, GLÄSNER J, REISCHL U, et al. Analyses of intestinal microbiota:culture versus sequencing[J]. ILAR J, 2015, 56(2):228-240.
[42]	BROWNE H P, FORSTER S C, ANONYE B O, et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation[J]. Nature, 2016, 533(7604):543-546.
[43]	MOOTE P E, ZAYTSOFF S J M, POLO R O, et al. Application of culturomics to characterize diverse anaerobic bacteria from the gastrointestinal tract of broiler chickens in relation to environmental reservoirs[J]. Can J Microbiol, 2020, 66(4):288-302.
[44]	龚艳清, 郭书林, 陈信忠, 等. 基质辅助激光解析电离飞行时间质谱在动物病原菌检测中的应用[J]. 中国动物检疫, 2012, 29(2):22-25.GONG Y Q, GUO S L, CHEN X Z, et al. Application of matrix -assisted laser desorption ionization-time of flight mass spectrometry in detection of animal pathogenic bacteria[J]. China Journal of Animal Quarantine, 2012, 29(2):22-25. (in Chinese)
[45]	DANZEISEN J L, KIM H B, ISAACSON R E, et al. Modulations of the chicken cecal microbiome and metagenome in response to anticoccidial and growth promoter treatment[J]. PLoS One, 2011, 6(11):e27949.
[46]	WANG L Q, LILBURN M, YU Z T. Intestinal microbiota of broiler chickens as affected by litter management regimens[J]. Front Microbiol, 2016, 7:593.
[47]	SKLAN D, SHACHAF B, BARON J, et al. Retrograde movement of digesta in the duodenum of the chick:extent, frequency, and nutritional implications[J]. J Nutr, 1978, 108(9):1485-1490.
[48]	STANLEY D, GEIER M S, DENMAN S E, et al. Identification of chicken intestinal microbiota correlated with the efficiency of energy extraction from feed[J]. Vet Microbiol, 2013, 164(1-2):85-92.
[49]	ZHAO L L, WANG G, SIEGEL P, et al. Quantitative genetic background of the host influences gut microbiomes in chickens[J]. Sci Rep, 2013, 3(1):1163.
[50]	SINGH P, KARIMI A, DEVENDRA K, et al. Influence of penicillin on microbial diversity of the cecal microbiota in broiler chickens[J]. Poult Sci, 2013, 92(1):272-276.
[51]	BORDA-MOLINA D, VITAL M, SOMMERFELD V, et al. Insights into broilers' gut microbiota fed with phosphorus, calcium, and phytase supplemented diets[J]. Front Microbiol, 2016, 7:2033.
[52]	SHANG Y, KUMAR S, OAKLEY B, et al. Chicken gut microbiota:importance and detection technology[J]. Front Vet Sci, 2018, 5:254.
[53]	BORDA-MOLINA D, SEIFERT J, CAMARINHA-SILVA A. Current perspectives of the chicken gastrointestinal tract and its microbiome[J]. Comput Struct Biotechnol J, 2018, 16:131-139.
[54]	CLAVIJO V, FLÓREZ M J V. The gastrointestinal microbiome and its association with the control of pathogens in broiler chicken production:a review[J]. Poult Sci, 2018, 97(3):1006-1021.
[55]	朱春红, 陶志云, 刘宏祥, 等. 家禽肠道菌群多样性及其与宿主的相互关系[J]. 安徽农业科学, 2018, 46(26):11-14.ZHU C H, TAO Z Y, LIU H X, et al. Diversity of intestinal microbiota in poultry and relationship with host[J]. Journal of Anhui Agricultural Sciences, 2018, 46(26):11-14. (in Chinese)
[56]	杨蕴涵, 张恩, 杨倩. 猪鼻腔内菌群特点及其鉴定[J]. 畜牧与兽医, 2018, 50(9):42-45.YANG Y H, ZHANG E, YANG Q. Characteristics and identification of bacterial floras inhabiting in the nasal cavity of pigs[J]. Animal Husbandry & Veterinary Medicine, 2018, 50(9):42-45. (in Chinese)
[57]	LOWE B A, MARSH T L, ISAACS-COSGROVE N, et al. Defining the "core microbiome" of the microbial communities in the tonsils of healthy pigs[J]. BMC Microbiol, 2012, 12(1):20.
[58]	KHANNA T, FRIENDSHIP R, DEWEY C, et al. Methicillin resistant Staphylococcus aureus colonization in pigs and pig farmers[J]. Vet Microbiol, 2008, 128(3-4):298-303.
[59]	VAN RIJEN M M L, BOSCH T, HECK M E O C, et al. Meticillin-resistant Staphylococcus aureus epidemiology and transmission in a Dutch hospital[J]. J Hosp Infect, 2009, 72(4):299-306.
[60]	KASPAR U, VON LVTZAU A, SCHLATTMANN A, et al. Zoonotic multidrug-resistant microorga-nisms among small companion animals in Germany[J]. PLoS One, 2018, 13(12):e0208364.
[61]	KASPAR U, VON LVTZAU A, SCHLATTMANN A, et al. Zoonotic multidrug-resistant microorga-nisms among non-hospitalized horses from Germany[J]. One Health, 2019, 7:100091.
[62]	CUNY C, FRIEDRICH A, KOZYTSKA S, et al. Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species[J]. Int J Med Microbiol, 2010, 300(2-3):109-117.
[63]	BECKER K, BALLHAUSEN B, KAHL B C, et al. The clinical impact of livestock-associated methicillin-resistant Staphylococcus aureus of the clonal complex 398 for humans[J]. Vet Microbiol, 2017, 200:33-38.
[64]	SCHLATTMANN A, VON LVTZAU K, KASPAR U, et al. The porcine nasal microbiota with particular attention to livestock-associated methicillin-resistant Staphylococcus aureus in Germany-a culturomic approach[J]. Microorganisms, 2020, 8(4):514.
[65]	STACKEBRANDT E, EBERS J. Taxonomic parameters revisited:tarnished gold standards[J]. Microbial Today, 2006, 33(4):152-155.
[66]	WEESE J S, SLIFIERZ M, JALALI M, et al. Evaluation of the nasal microbiota in slaughter-age pigs and the impact on nasal methicillin-resistant Staphylococcus aureus (MRSA) carriage[J]. BMC Vet Res, 2014, 10(1):69.
[67]	STRUBE M L, HANSEN J E, RASMUSSEN S, et al. A detailed investigation of the porcine skin and nose microbiome using universal and Staphylococcus specific primers[J]. Sci Rep, 2018, 8(1):12751.
[68]	KASPAR U, KRIEGESKORTE A, SCHUBERT T, et al. The culturome of the human nose habitats reveals individual bacterial fingerprint patterns[J]. Environ Microbiol, 2016, 18(7):2130-2142.
[69]	万遂如. 养猪生产中如何控制动物源细菌耐药性与兽药残留[J]. 养猪, 2020(3):1-3.WAN S R. How to control the drug resistance and veterinary drug residues of bacteria from animals in swine production[J]. Swine Production, 2020(3):1-3. (in Chinese)
[70]	孙康泰, 张建民, 蒋大伟, 等. 我国动物源细菌耐药性的研究进展及防控策略[J]. 中国农业科技导报, 2020, 22(5):1-5.SUN K T, ZHANG J M, JIANG D W, et al. Progress and countermeasures of antimicrobial resistance of animal origin bacterial pathogens in China[J]. Journal of Agricultural Science and Technology, 2020, 22(5):1-5. (in Chinese)

The Application of Microbiome Culturomics in Veterinary Medicine

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