The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 × 10⁵ cfu/g of E. coli O157:H7 (EC); (3) EC and 1 × 10⁶ cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 × 10⁶ cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 ± 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 ± 3.80%). Relative to the control, adding E. coli O157:H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = −0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = −0.51), Pantoea (r = −0.46), Pseudomonas (r = −0.45), and Stenotrophomonas (r = −0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = −0.59), Sphingomonas (r = −0.66), Pantoea (r = −0.69), Pseudomonas (r = −0.69), and Stenotrophomonas (r = −0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.

这项研究的第一个目的是研究添加或不添加化学或微生物添加剂的大肠杆菌O157：H7对苜蓿青贮饲料细菌多样性和组成的影响。第二个目标是检查已知和未知细菌种类的相对丰度与青贮饲料发酵质量指标之间的关联。收获苜蓿草料，干物质含量为54％，切碎至理论切割长度19 mm，并在进行以下处理后一式四份地在实验室筒仓中青贮100 d：（1）蒸馏水（对照）；（2）1×10 ⁵ cfu / g大肠杆菌O157：H7（EC）; （3）EC和1×10 ⁶ cfu / g植物乳杆菌（EC + LP）；（4）EC和1×10 ⁶ cfu / g布氏乳杆菌（EC + LB）；（5）EC和0.22％的丙酸（EC + PA）。放置100天后，通过Illumina MiSeq平台（Illumina Inc.，圣地亚哥，加利福尼亚）分析青贮饲料样品的细菌多样性和组成，并进行化学表征。总体而言，Firmicutes（74.1±4.86％）是最主要的门类，其次是Proteobacteria（20.4±3.80％）。相对于对照，在培养过程中单独添加大肠杆菌O157：H7不会影响细菌多样性或组成，但是添加EC + LP或EC + LB会降低Shannon指数（衡量多样性的指标）（分别为3.21和2.63或2.80）。 。沙门氏菌的相对丰度与对照组（79.5和16.5％）和EC + LB（77.4和77.4）相比，EC + LP和EC + PA处理降低了（69.2和68.8％）变形杆菌的细菌（24.0和24.9％）。青贮分别为18.5％）。与对照组相比，EC + LP处理分别使乳杆菌，鞘氨醇单胞菌，泛菌，假单胞菌和欧文氏菌的相对丰度分别提高了426％，157％，200％，194％和163％，但降低了Pediococcus，Weissella和Methylobacterium的相对丰度。分别减少了5,436、763和250％。Weissella（9.19％）和甲基杆菌的相对丰度与对照组相比，EC + LB青贮饲料的含量也降低了（0.94％）（分别为29.7和1.50％）。丙酸的施用不会影响乳酸杆菌，魏氏杆菌或小球菌的相对丰度。乳酸浓度呈正相关与相对丰度（R = 0.56）乳杆菌和负相关（r = -0.41），用相对丰度片球菌属。氨氮浓度与鞘氨醇单胞菌（r = -0.51），泛菌（r = -0.46），假单胞菌（r = -0.45）和嗜麦芽单单胞菌的相对丰度之间呈负相关。（r ＝ -0.38）。青贮饲料的pH值与乳酸杆菌（r = -0.59），鞘氨醇单胞菌（r = -0.66），泛菌（r = -0.69），假单胞菌（r = -0.69）和嗜麦芽单胞菌（r = -0.50）的相对丰度呈负相关。未来的研究应旨在鉴定，培养和确定本研究中检测到的未知细菌的功能，以阐明其在青贮饲料发酵中的作用。