The Cultured Microbiota Analysis Of Tibetan Antelope Feces And Plateau Pika Intestinal Content And Discovery Of Novel Bacterial Species

Objective:Tibetan antelopes and Plateau pikas are the endemic animals of the Qinghai-Tibet Plateau,living in cold,hypoxic areas with high altitude,and carrying many unknown microorganisms.This study aims to systematically analyze the cultured bacterial flora of Tibetan antelope feces and intestine contents of Plateau pika,isolate and discover novel species,and explore their possible biological implications.Methods:1.The colonies were isolated and purified after culture on different agars?including brain heart infusion containing sheep blood agar,TSA agar,NA agar,etc.?at 28/37?in two gas environments?aerobic and 5%CO₂?.The 16S rRNA gene sequence was obtained by PCR amplification and Sanger sequencing after extraction of genomic nucleic acid.The similarity between the 16S rRNA gene sequence of strains and the known species was obtained through GenBank and EzTaxon databases.2.For those strains identified as suspected novel species,the basic characteristics were examined through Gram staining,transmission electron microscopy,peacock blue staining,and kinetic experiments.Phylogenetic tree analysis was performed based on 16S rRNA gene sequences and genomic sequences.The physiological and biochemical characteristics were examined through sugar fermentation,enzyme activity test and antibiotics sensitivity test,etc.The bacterial cell compositions were examined via Reversed-phase high-pressure liquid chromatography,two-dimensional thin-layer chromatography,and HITACHI L-8900 high-speed amino acid analyzer.Genome was analyzed through genomic hybridization and annotation.The taxonomic location of strains was determined based on the analyze of phenotype,genotype and biochemical characteristics.The novel species were named,and the type strains were preservation.For those strains identified as known species,the diversity and medical significance were analyzed.Results:1.Under 2 identical culture conditions,525 strains were successfully identified from the intestinal contents of pikas,belonging to 3 bacteria phyla,6 bacteria class,12 bacteria orders,20 bacteria families,23 genera,52 species.Meanwhile,734 strains were successfully identified from the feces of Tibetan antelopes,belonging to 3 phylums,4bacterae,9 bacteria orders,20 bacteria families,28 genera and 73 species.Under 22culture conditions,1977 strains were successfully identified from Tibetan antelope feces,belonging to 4 phyla,7 families,15 orders,30 families,44 genera and 123 species.Twenty-three of 123 species were isolated from clinical patients,including Acinetobacter schindleriis,Actinomyces johnsonii,Bacillus circulans,B.licheniformis,B.mojavensis,B.pumilus,B.safensis,B.thuringiensis,Clostridium perfringens,Corynebacterium lipophiloflavum,Dietzia maris,D.natronolimnaea,Enterococcus durans,E.faecium,E.mundtii,E.gallinarum,Kocuria rosea,Oerskovia turbata,Pseudomonas oryzihabitans,Staphylococcus cohnii,S.equorum,S.warneri and S.hominis subsp.novobiosepticus.The dominant species isolated from Tibetan antelope feces were Arthrobacter citreus,Paenibacillus xylanexedens and Enterococcus faecium.2.Seven novel species were isolated from Tibetan antelope feces,numbered as 449^T,194^T,2129^T,2251^T,2183^T,2184^T and 592^T.?1?Strain 449^T is a novel species of Mycetocola genus,named as Mycetocola zhujimingii.The cells contained anteiso-C_15:05:0 as the major cellular fatty acid,MK-10 and MK-11 as predominant menaquinones,diphosphatidylglycerol and phosphatidylglycerol as major polar lipids,and lysine as the diagnostic diamino acid.In the phylogenetic tree,the strain was closest to M.zhadangensis.?2?Strain 194^T is a novel species of Salinibacterium genus,named as Salinibacterium hongtaonis.The strain was able to grow in medium up to 10%NaCl.The strain had anteiso-C_15:05:0 and anteiso-C_17:07:0 as the major fatty acids,and their cell walls contained lysine,alanine,glycine and glutamic acid.The predominant menaquinones identified were MK-11 and MK-10,with diphosphatidylglycerol and phosphatidylglycerol as the major polar lipids.Strain 194^T was unable to utilize D-mannose or produce naphthol-AS-BI-phosphohydrolase.?3?Strain 2129^T was named as Actinomyces lilanjuaniae.The major fatty acids of the strain were C_18:1?9c and C_16:0,and the major polar lipids were diphosphatidylglycerol,glycolipid,phosphatidylinositol,phosphatidyl inositol mannoside and phosphoglycolipid.Strain 2129^T could ferment D-adonitol and D-xylose,but was unable to utilize D-mannose and D-melibiose or produce esterase?C4?and proline arylamidase.In the phylogenetic tree,the strain was closest to A.dentalis and A.slackii.?4?Strain 2251^T was named as Paracoccus liaowanqingii,contained C_18:1?7c as the major cellular fatty acid and ubiquinone-10 as the predominant menaquinone.The polar lipids comprised phosphatidylethanolamine,diphosphatidylglycerol,phosphatidylglycerol,phospholipid,glycolipid and an unidentified lipid.The cell wall peptidoglycan amino acids were meso-2,6-diaminopimelic acid,alanine and glutamic acid.Phylogenetic analysis showed that strain 2251^T was closest to P.gahaiensis.?5?Strains 2183^T and 2184^T were named as Corynebacterium yudongzhengii and Corynebacterium liangguodongii,respectively.The two strains were nitrate reduction negative,?-chymotrypsin and catalase positive,and produced acid from D-fructose and 5-potassium ketogluconate but not from D-lactose.Bothstrainscontaineddiphosphatidylglycerol,phosphatidylglycerol,phosphatidylinositol and phosphatidylinositol-mannoside as the major polar lipids,MK-8?H₂?and MK-9?H₂?as the major respiratory quinones,and meso-2,6-diaminopimelic acid in the peptidoglycan.In the phylogenetic tree,C.yudongzhengii was closest to C.doosanense and C.tapiri,while C.liangguodongii was closest to C.lipophiloflavum and C.mycetoides.?6?Strain 592^T was named as Aeromicrobium chenweiae,producing acid from L-rhamnose,D-xylose,D-cellobiose,but being unable to reduce nitrate.The major fatty acids of the strain were C_18:1?9c and C_18:010-methyl,and the main polar lipids were diphosphatidylglycerol,phosphatidylglycerol and phosphatidylinositol.The predominant respiratory quinones were MK-9?H₄?and MK-8?H₄?.The cell wall peptidoglycan contained LL-diaminopimelic acid.3.Two novel species were isolated from intestinal content of Plateau pikas,numbered as MF30-A^T and MF47^T.?1?Strain MF-30A^T was named as Agromyces badenianii,being aerobic and Gram-stain-positive bacteria.Optimal growth of the two strain was observed under aerobic condition at pH 7.0 and 28?.The strain could not produce acid from arbutin,D-fructose,D-sucrose,glycogen,salicin or starch.Production of?-glucosidase by these strains was negative.In the polygenetic tree,strain MF30-A^T clearly distinct from other species.?2?Strain MF47^T was named as Aeromicrobium yanjiei,being distinct from A.chenweiae.The strain produced acid from L-rhamnose,D-xylose,D-cellobiose,but was unable to reduce nitrate.The major fatty acids of the strain were C_18:1?9c and C_18:08:0 10-methyl,and the main polar lipids were diphosphatidylglycerol,phosphatidylglycerol and phosphatidylinositol.The predominant respiratory quinones were MK-9?H₄?and MK-8?H₄?.The cell wall peptidoglycan contained LL-diaminopimelic acid.Conclusion:1.Under 2 identical culture conditions,525 strains were successfully identified from the intestinal contents of pikas,belonging to 3 bacteria phyla,6 bacteria class,12 bacteria orders,20 bacteria families,23 genera,52 species.Meanwhile,734 strains were successfully identified from the feces of Tibetan antelopes,belonging to 3 phylums,4bacterae,9 bacteria orders,20 bacteria families,28 genera and 73 species.1977 strains were successfully identified from Tibetan antelope feces,belonging to 4 phyla,7 families,15 orders,30 families,44 genus and 123 species.Twenty-three of 123 species have been isolated from clinical samples with potential medical significance.2.Nine suspected novel species were isolated from feces of Tibetan antelope and intestinal contents of Plateau pika on the Qinghai-Tibet Plateau.Based on the phenotype detection,genomic analysis,biochemical characteristics and cell composition,the nine suspected novel bacteria were identified as novel species.In order to commemorate the contributions of certain academicians and researchers,combined with the international naming rules of novel species,the nine novel species were named as Mycetocola zhujimingii,Salinibacterium hongtaonis,Actinomyces lilanjuaniae,Paracoccus liaowanqingii,Agromyces badenianii,Corynebacterium yudongzhengii,Corynebacterium liangguodongii,Aeromicrobium chenweiae and Aeromicrobium yanjiei,respectively.Five of nine novel species had been published,and the other four species had been submitted for consideration.