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Published on: August 12, 2020
Reducing Viability Bias in Analysis of Gut Microbiota in Preterm Infants at Risk of NEC and Sepsis
Gregory R Young1, Darren L Smith1, Nicholas D Embleton2
1Faculty of Health and Life Sciences, University of NorthumbriaNewcastle upon Tyne, United Kingdom.
Insights
Propidium monoazide (PMA) treatment accurately identifies viable gut bacteria in preterm infants, reducing bias in analyses of necrotising enterocolitis (NEC) and sepsis risk. This method improves understanding of the preterm gut microbiota
Area of Science:
- Microbiology
- Neonatal Medicine
- Genomics
Background:
- Necrotising enterocolitis (NEC) and sepsis are critical conditions in preterm infants with high mortality rates.
- Improvements in neonatal care increase survival but also the number of infants at risk for NEC and sepsis.
- The role of gut microbiota in NEC pathogenesis is not fully understood, partly due to methodological limitations in identifying viable bacteria.
Purpose of the Study:
- To validate the use of propidium monoazide (PMA) in conjunction with 16S rRNA gene sequencing to accurately characterize the viable gut microbiota in preterm infants.
- To reduce bias in microbiota analysis caused by non-viable bacterial DNA, thereby improving the accuracy of clinical inferences.
- To investigate the impact of PMA treatment on the diversity and composition of the gut microbiota in relation to NEC and sepsis risk.
Main Methods:
- Clinical stool samples from 16 matched preterm infant twins who received probiotics were analyzed.
- Samples were treated with propidium monoazide (PMA), a DNA chelating agent, before bacterial DNA extraction and 16S rRNA gene sequencing.
- Meta-analysis was performed to compare microbiota profiles between PMA-treated and untreated samples.
Main Results:
- PMA treatment significantly reduced bacterial diversity in 68.8% of samples and decreased the abundance of rare taxa.
- Abundances of key bacterial genera, including Bifidobacterium, Clostridium, and Staphylococcus, known to be associated with NEC and sepsis, showed significant differences after PMA treatment.
- Exclusion of non-viable cells by PMA treatment altered the perceived composition of the gut microbiota.
Conclusions:
- Propidium monoazide (PMA) treatment effectively differentiates viable from non-viable bacteria in preterm infant stool samples, reducing bias in 16S rRNA gene sequencing.
- This improved methodology enhances the accuracy of clinical inferences regarding the preterm gut microbiota's role in infant health and disease, specifically NEC and sepsis susceptibility.
- The study highlights the importance of assessing viable microbiota for a more precise understanding of infant gut microbiome dynamics and its clinical implications.
Abstract:
Necrotising enterocolitis (NEC) and sepsis are serious diseases of preterm infants that can result in feeding intolerance, the need for bowel resection, impaired physiological and neurological development, and high mortality rates. Neonatal healthcare improvements have allowed greater survival rates in preterm infants leading to increased numbers at risk of developing NEC and sepsis. Gut bacteria play a role in protection from or propensity to these conditions and have therefore, been studied extensively using targeted 16S rRNA gene sequencing methods. However, exact epidemiology of these conditions remain unknown and the role of the gut microbiota in NEC remains enigmatic. Many studies have confounding variables such as differing clinical intervention strategies or major methodological issues such as the inability of 16S rRNA gene sequencing methods to determine viable from non-viable taxa. Identification of viable community members is important to identify links between the microbiota and disease in the highly unstable preterm infant gut. This is especially important as remnant DNA is robust and persists in the sampling environment following cell death. Chelation of such DNA prevents downstream amplification and inclusion in microbiota characterisation. This study validates use of propidium monoazide (PMA), a DNA chelating agent that is excluded by an undamaged bacterial membrane, to reduce bias associated with 16S rRNA gene analysis of clinical stool samples. We aim to improve identification of the viable microbiota in order to increase the accuracy of clinical inferences made regarding the impact of the preterm gut microbiota on health and disease. Gut microbiota analysis was completed on stools from matched twins (n = 16) that received probiotics. Samples were treated with PMA, prior to bacterial DNA extraction. Meta-analysis highlighted a significant reduction in bacterial diversity in 68.8% of PMA treated samples as well as significantly reduced overall rare taxa abundance. Importantly, overall abundances of genera associated with protection from and propensity to NEC and sepsis such as: Bifidobacterium; Clostridium, and Staphylococcus sp. were significantly different following PMA-treatment. These results suggest non-viable cell exclusion by PMA-treatment reduces bias in gut microbiota analysis from which clinical inferences regarding patient susceptibility to NEC and sepsis are made.
