Lysozyme incubation affects the total bacteria content extracted from rectum
qPCR was used to test if the total bacteria content in rectum can be affected by lysozyme incubation. The results showed that total bacteria content could be significantly affected by lysozyme incubation. For newly emerged male, bacteria content in rectum was significantly higher if the DNA was extracted without lysozyme treatment. However, bacteria contents were significantly lower if the DNA was extracted without lysozyme treatment in 3- and 6-day-old male. For 9- and 12-day-old male, there was no difference for total bacteria contents (Figure 1).
Rectum bacteria diversity was significantly affected by lysozyme incubation
To test if lysozyme incubation would affect bacteria diversity, 16S rRNA amplicon sequencing was used to investigate rectum bacteria diversity. We assessed the impact of lysozyme incubation on sequencing data. The results showed that both sequencing tag and OTU numbers were significantly higher in lysozyme incubation samples (Figure 2A and 2B). Furthermore, α diversity and β diversity represented by Shannon indices and PCoA analysis were assessed. The results were in agreement with the sequencing data evaluation results: Shannon indices were significantly higher in lysozyme incubation samples (Figure 2C). PCoA plots of non-lysozyme treatment samples showed that the rectum samples collected at different time points were highly clustered, while distinct separations were showed in lysozyme treatment samples (Figure 2D and 2E). These results indicate lysozyme treatment before DNA extraction is vital for investigating the bacteria diversity in rectum accurately.
Lysozyme treatment significantly affected the taxonomic results
Taxonomic compositions of the rectum bacteria were compared to test whether lysozyme treatment can affect the bacteria community composition. For lysozyme treated samples, rectum bacteria changed significantly at different time points. The dominant taxa in 0d samples was Gammaproteobacteria and Bacilli, while Bacilli was the only dominant taxa in 3d, 6d, 9d, and 12d samples. However, bacteria composition patterns were significantly different in non-lysozyme treated samples. Gammaproteobacteria, Bacilli and Clostridia were the dominant taxa in rectum at all the time points and the abundance of the same bacteria group fluctuated greatly in the repeated samples, which indicated that DNA extraction efficiency of the rectum bacteria was very unstable (Figure 3).
Since lysozyme can effectively improve DNA extraction efficiency of Gram-positive bacteria , We divided the rectum bacteria into two groups (Gram-negative and Gram-positive bacteria) and compared their abundance. In the top six groups in term of abundance, Bacilli, Clostridia and Actinobacteria were mainly Gram-positive bacteria, while Gammaproteobacteria, Bacteroidia and Alphaproteobacteria were mainly Gram-negative bacteria (Figure 3). For 0d rectum samples, none of Gram-positive and Gram-negative bacteria were significantly different in abundance between samples treated with lysozyme and control (Figure 4). However, Gram-positive bacteria abundance were significantly higher in samples treated with lysozyme for 3d, 6d, 9d and 12d rectum and the abundance of Gram-negative bacteria were significantly lower (Figure 4). These results indicated that lysozyme can significantly increase the abundance of Gram-positive bacteria extracted in rectum.