Abstract
Gastrointestinal bacteria are essential for host health, and only viable microorganisms contribute to gastrointestinal functions. When evaluating the gut microbiota by next generation sequencing method, dead bacteria, which compose a proportion of gut bacteria, may distort analysis of the live gut microbiota. We collected stomach, jejunum, ileum, cecum, and colon contents from Rex rabbits. A modified propidium monoazide (PMA) treatment protocol was used to exclude DNA from dead bacteria. Analysis of untreated samples yielded total bacteria, and analysis of PMA-treated samples yielded live bacteria. Quantitative polymerase chain reaction and 16S rRNA gene sequencing were performed to evaluate the live-to-total bacteria ratio and compare the difference between live and total microbiota in the entire digestive tract. A low proportion of live bacteria in the foregut (stomach 1.12%, jejunum 1.2%, ileum 2.84%) and a high proportion of live bacteria in the hindgut (cecum 24.66%, colon 19.08%) were observed. A significant difference existed between total and live microbiota. Clostridiales, Ruminococcaceae, and S24-7 dominated the hindgut of both groups, while Acinetobacter and Cupriavidus dominated only in live foregut microbiota. Clostridiales and Ruminococcaceae abundance decreased, while S24-7 increased in live hindgut microbiota. The alpha- and beta-diversities differed significantly between groups. Analysis of networks showed the mutual relationship between live bacteria differed vastly when compared with total bacteria. Our study revealed a large number of dead bacteria existed in the digestive tract of Rex rabbits and distorted the community profile of the live microbiota. Total bacteria is an improper representation of the live gut microbiota, particularly in the foregut.
Highlights
Gastrointestinal microbiota is essential for host health and nutrient utilization, and only viable microorganisms are capable of contributing to these functions
propidium monoazide (PMA) treatment induced a significant decrease in dead bacteria as compared with that in non-PMA-treated samples
Comparing the profiles of live and total bacteria, a change in the ranking of most abundant phyla in the foregut was observed. In the hindgut these changes were less severe, as the relative abundance of Firmicutes and Bacteroidetes changed only slightly. These findings demonstrate that dead bacteria are a more substantial interference to the foregut microbiota
Summary
Gastrointestinal microbiota is essential for host health and nutrient utilization, and only viable microorganisms are capable of contributing to these functions. 16S rRNA gene high-throughput sequencing has the ability to more completely evaluate microbiota complexity (Medini et al, 2008), it cannot discriminate between live and dead gut bacteria. Propidium monoazide (PMA) only penetrates membranedamaged cells, where a photo-induced azide group covalently binds to DNA. This cross-linking effectively inhibits PCR amplification of DNA from dead cells (Nocker et al, 2006; Bae and Wuertz, 2012) of both Gram-negative and Gram-positive bacteria (Nocker and Camper, 2009). High bacterial concentrations (beyond 8 log/ml) and high concentrations of opaque impurities reduce light transparency, decreasing the efficacy of PMA-mediated exclusion of dead bacteria (Wagner et al, 2008; Nocker and Camper, 2009; Frankenhuyzen et al, 2011), which, until now, has impeded the application of PMA to gut samples
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