| Caves are subsurface space and tunnel formed by the dissolution of rocks or during the cooling process of lava.Caves constitute oligotrophic ecosystems characterized by total darkness or lowlevel of light,low stable temperature and high humidity,but maintain highly microbiomes.With the cultivating and culture-independent methods to explore the bacterial diversity of Ji Guan dong(JGD)and its neighbouring unnamed cave,Wu Ming dong(WMD)and revealed their bacterial diversity and community compositions in these two caves.The two potential new species were identified with a polyphasic taxonomy method.Finally,preliminary screening of antibacterial activity of the isolated strains was carried out against the pathogenic bacteria of aquatic animals to determine the bioactive strains.Through the high-throughput sequencing,14 samples collected from the two caves revealed 224,109draft sequences and annotated to 1,800 OTUs with an average length of 294 bp.These OTUs were affiliated to 36 phyla,89 classes,123 orders,136 families and 172 genera.The predominant bacterial phyla were Proteobacteria(37.3%),Actinobacteria(18.7%),Chloroflexi(8.2%),Acidobacteria(7.1%),Bacter-oidetes(5.7%),and Planctomycetes(5.6%).Using a series of isolation media(10)and two different temperatures,a total of 1,071 strains were isolated.After dereplication and 16S rRNA gene sequencing,the classification information of 676 strains was obtained,and the result revealed that these strains were distributed into four phyla(Actinobacteria,Proteobacteria,Firmicutes and Deinococcus-Thermus),8 classes,20 orders,37 families,70 genera.The phylum Actinobacteria was the most widely distributed microbial group in the cave samples,with a total of 530 strains.The genera Nocardioides(30.75%)and Streptomyces(16.98%)were the mostabundant at the generic level.This was followed by isolates affiliated to the phyla Firmicutes(111 strains)and Proteobacteria(35 strains).The cultivated microbial community structure between the two caves was different.In JGD cave,the dominant microbial groups were Nocardioides,Streptomyces,Microbacterium,Bacillus,Pseudonocardia,and Nocardia;but in WMD cave,the dominant groupwas Bacillus,Rhodococcus,Arthrobacter,and Streptomyces.Additionally,the species richness at 28? was found higher than that at 15? as revealed by the higher number of genera isolated at 28?(37 genera)than in 15?(33 genera)in the JGD samples.The two novel species Ornithinimicrobium cavernae sp.nov.and Nocardioides allogilvus sp.nov.were identified by the polyphasic taxonomy method from among these isolates.To further screen the strains with inhibitory effects on pathogenic bacteria,49 isolated were selected from among the strains isolated in the study.These strains were cultured in three fermentation media RA,ISP 2 and AM3,and the crude fermentation extracts tested for inhibition of eight pathogenic bacteria.The assay showed that the 26 tested strains had inhibitory effect on more than 6 indicator strains,accounting for 59.2%of all strains;and had a best bacteriostatic effect on Vibrio parahaemolyticus.Among 147 fermentation products from 49 strains,133 fermentation products had antibacterial effect on Aeromonas hydrophila(account for 90.5%),129 fermentation products had antibacterial effect on Vibrio parahaemolyticus(account for 87.8%)and 116 fermentation products had inhibitory effect on Methicillin-resistant Staphyococcus aureus(account for 78.9%). |
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