Systematic Evaluation of the Viable Microbiome in the Human Oral and Gut Samples with Spike-in Gram+/– Bacteria

• DOI: 10.1128/msystems.00738-22
• Publication Date: 2023-03-27T14:01:40Z
• Authors: Feng Liu; Hui Lu; Biao Dong; Xiaochang Huang; Hongyu Cheng; Ru Qu; Yichen Hu; Luyun Zhong; Zhenni Guo; Yuehua You; Zhenjiang Zech Xu
• Keywords: Gram

PMA (propidium monoazide) is one of the few methods that are compatible with metagenomic sequencing to characterize live/intact microbiota. However, its efficiency in complex communities such as saliva and feces still controversial. An effective method for depleting host dead bacterial DNA human microbiome samples lacking. Here, we systematically evaluate osmotic lysis PMAxx treatment (lyPMAxx) characterizing viable four live/dead Gram+/Gram- microbial strains simple synthetic spiked-in communities. We show lyPMAxx-quantitative PCR (qPCR)/sequencing eliminated more than 95% heat-killed had a much smaller effect on live microbes both mock The overall load alpha diversity salivary fecal were decreased by lyPMAxx, relative abundances changed. Actinobacteria, Fusobacteria, Firmicutes was feces. also found frequently used sample storage method, freezing glycerol, killed or injured 65% 94% living cells feces, respectively, Proteobacteria phylum affected most Bacteroidetes phyla By comparing absolute abundance variation shared species among different types individuals, habitat personal differences response lyPMAxx freezing. IMPORTANCE functions phenotypes largely defined microbes. Through advanced nucleic acid technologies downstream bioinformatic analyses, gained an insight into high-resolution community composition yet know very little about whether sequences represent PMA-qPCR previous studies. spiking-in strains, demonstrate can effectively discriminate between (saliva feces). In addition, kill injure significantly, measured lyPMAxx-qPCR/sequencing. This has promising prospect viable/intact microbiota detection