Gut microbiota
PCR amplification from bronchoalveolar lavage fluid and fecal samples was performed on the V3-V4 region (hypervariable region) of bacterial 16S rDNA (Forward: ACTCCTACGGGAGGCAGCA, Reverse: GGACTACHVGGGTWTCTAAT) was amplified via PCR. Then the PCR amplification products were collected by cutting and quantified using a QuantiFluorTM fluorescence meter. Mix the purified PCR amplification products in equimolar amounts, ligate sequencing adapters, and construct a sequencing library for Illumina PE250 sequencing. Following high-throughput sequencing, paired-end reads were quality-filtered, merged into contiguous sequences, chimeric sequences were identified and removed using UCHIME or VSEARCH, and operational taxonomic units (OTUs) were clustered at 97% sequence identity using UPARSE. Their relative abundances were quantified to enable downstream microbial community analysis. The most abundant sequence within each OTU was selected as its representative sequence. Alpha diversity indices (Chao1, ACE, Shannon, Simpson) were calculated for samples from the infected group and blank control group and visualized with R. LEfSe analysis was performed via Omicsmart online system (https://www.omicsmart.com/home.html#/project). Beta diversity was assessed using Omicsmart online system, including principal component analysis (PCA) and nonmetric multidimensional scaling (NMDS), primarily to reveal differences in microbial community structure between samples across groups. Results were visualized using R software, with |r| > 0.4 and P < 0.05 as the threshold for significant correlation.