Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 14, 2017; 23(42): 7584-7593
Published online Nov 14, 2017. doi: 10.3748/wjg.v23.i42.7584
Diversity of bacterial lactase genes in intestinal contents of mice with antibiotics-induced diarrhea
Cheng-Xing Long, Lu He, Yan-Fang Guo, Ya-Wei Liu, Nen-Qun Xiao, Zhou-Jin Tan
Cheng-Xing Long, Lu-He, Yan-Fang Guo, Ya-Wei Liu, Nen-Qun Xiao, Zhou-Jin Tan, Department of Microbiology, Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
Cheng-Xing Long, College of Mathematics and Finance, Hunan University of Humanities, Science and Technology, Loudi 417000, Hunan Province, China
Author contributions: Tan ZJ designed the study; He L and Guo YF performed the experiments; Liu YW and Long CX analyzed the data; Long CX and Xiao NQ wrote the paper.
Supported by the National Natural Science Foundation of China, No. 81573951.
Institutional review board statement: The study was approved by the Institutional Review Board of Hunan University of Chinese Medicine, Changsha, China.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Animal Ethics and Welfare Committee of Hunan University of Chinese Medicine, Animal License SCXK (Xiang) 2013-0004.
Conflict-of-interest statement: The authors declare no conflict of interest related to this article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Zhou-Jin Tan, PhD, Professor, Department of Microbiology, Hunan University of Chinese Medicine, 300 Xueshi Road, Changsha 410208, Hunan Province, China. tanzhjin@sohu.com
Telephone: +86-13974954942
Received: July 16, 2017
Peer-review started: July 18, 2017
First decision: August 30, 2017
Revised: September 14, 2017
Accepted: September 26, 2017
Article in press: September 26, 2017
Published online: November 14, 2017
Processing time: 118 Days and 17.2 Hours
ARTICLE HIGHLIGHTS
Research background

Studies have shown that drug- or antibiotics-induced diarrhea is associated with intestinal lactase dysfunction due to loss of activity. Thus, treatment with lactase supplements is a good option for most types of diarrhea due to the importance of lactase activity in the control of intestinal function. Various isoforms of the lactase gene have been identified and are widely expressed in the intestinal tract, with diversity enzyme activities. The expression, protein modification and isoforms can change in different micro-environments. Antibiotics-associated diarrhea is not only associated with dysbacteriosis but also intestinal lactase activity damage, leading to diarrhea. In the present study, we found that the activity of lactase in intestinal contents was significantly reduced in mice with antibiotics-induced diarrhea.

Research motivation

The mechanism of antibiotics-induced diarrhea has been studied in a wide range of diverse microbes. However, less research has been carried out on functional enzymes. In our preliminary study, we found that the activity of lactase in intestinal contents was significantly reduced in mice with antibiotics-induced diarrhea. The present study was conducted in order to determine the mechanism of lactase activity from the viewpoint of genetic diversity and provide a basis for antibiotics-induced diarrhea.

Research objectives

This study was carried out in order to provide a basis for the mechanism of antibiotics-induced diarrhea and to determine whether the alterations in activity were caused by its expression. We compared the diversity of bacterial lactase genes expressed in model mice with antibiotics-induced diarrhea and in control mice.

Research methods

Twelve mature specific pathogen-free Kunming mice were randomly allocated to the control and model groups, with six mice in each group. The mouse model of antibiotics-induced diarrhea was created by gastric perfusion with mixed antibiotics (23.33 mL·kg-1·d-1) composed of gentamicin sulfate and cephradine capsules administered for 5 days; the control group received an equal amount of sterile water. The contents of the jejunum and the ileum were then collected and metagenomic DNA was extracted, followed by analysis of bacterial lactase genes using operational taxonomic units after amplification and sequencing. Qiime software was used to align the sequencing results and carry out cluster analysis, principal component analysis (PCA), ACE abundance indexing and Simpson diversity indexing analysis. Principal coordinates analysis (PCoA), nonmetric multidimensional scaling (NMDS) and heatmap analysis were carried out in the R for diversity and similarity. SPSS21.0 software was used for statistical analysis and the results are expressed as means ± SE.

Research results

The results showed that there were significant differences in Chao1 and ACE indices between the two groups (P < 0.05). As shown by PCA, PCoA and NMDS analysis, sample distribution in the control group was relatively intensive and differences among individuals were small, while in the model group, they were dispersed and more diversified. The bacterial lactase genes in the intestinal contents from the control mice were related to Proteobacteria, Actinobacteria, Firmicutes and unclassified bacteria. Of these, Proteobacteria showed the greatest abundance. In contrast, the bacterial population was less diversified and abundant in model mice, as the abundance of Bradyrhizobium sp. BTAi1, Agrobacterium sp. H13-3, Acidovorax sp. KKS102, Azoarcus sp. KH32C and Aeromonas caviae was lower than that in the control group. In addition, of the known species, the control group and model group had their own unique genera, respectively. For example, Gordonia, Mycobacterium, Frankia, Microbacterium, Novosphingobium and Aeromonas were only seen in the control group. However, Micromonospora, Paenibacillus and Ensifer were only found in the model group. To confirm our findings, the diversity and abundance of bacterial species were clearly shown using heatmap analysis. The lactase genes of Gordonia, Frankia, Novosphingobium, Mycobacterium, Agrobacterium, Aeromonas and Microbacterium were highly present in the intestinal contents from the model group compared with the control group.

Research conclusions

Antibiotics mainly changed the number of the lactase-producing strains or reduced the number of key lactase strains. Antibiotics reduce the diversity of the intestinal bacterial flora, change the lactase gene strains, and transform their structures.

Antibiotics-induced diarrhea reduced the diversity of bacterial lactase genes in the intestinal contents and decreased lactase activity by altering the number of lactase-producing strains or reducing the number of key lactase strains.

The activity of lactase in intestinal contents was significantly reduced in mice with antibiotics-induced diarrhea. The new hypotheses that this study proposed involves how to screen and identify certain key lactase-producing strains in intestinal contents.

The bacterial lactase gene primers were designed to analyze the diversity of bacterial lactase genes in intestinal contents of mice with antibiotics-induced diarrhea by PCR, gene diversity analysis and bioinformatics techniques.

There were significant differences between control group and model group mice shown by PCR, PCoA and NMDS analysis. Lactase from different bacterial sources has different nature and activity. The diversity of lactase-producing bacteria leads to diversity of lactase genes and their activities.

Antibiotics-induced diarrhea reduced the diversity of bacterial lactase genes in the intestinal contents and decreased lactase activity by altering the number of lactase-producing strains or reducing the number of key lactase strains, leading to diarrhea.

Research perspectives

Lactase genes are good indicators for determining the diversity of bacteria in intestinal contents, and may be used to monitor the health of mice by comparing the abundance and diversity of bacterial lactase gene expression. The lactase genes will help to explore the regulation mechanism of traditional Chinese medicine on intestinal lactase activity based on the relationship between intestinal lactase diversity and antibiotics-induced diarrhea. The best methods for future research are enzyme technology and gene diversity analysis technology.