Development and application of high throughput system for processing fecal samples for microbiota analysis : pilot study of prevalence of Lactobacillus rhamnosus GG in infants and mother-infant pairs

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Title: Development and application of high throughput system for processing fecal samples for microbiota analysis : pilot study of prevalence of Lactobacillus rhamnosus GG in infants and mother-infant pairs
Author: Koirala, Prabin
Other contributor: Helsingin yliopisto, Maatalous-metsätieteellinen tiedekunta, Elintarvike- ja ympäristötieteiden laitos
University of Helsinki, Faculty of Agriculture and Forestry, Department of Food and Environmental Sciences
Helsingfors universitet, Agrikultur- och forstvetenskapliga fakulteten, Institutionen för livsmedels- och miljövetenskaper
Publisher: Helsingin yliopisto
Date: 2018
Language: eng
Thesis level: master's thesis
Discipline: Biotekniikka (EYT)
Biotechnology (EYT)
Bioteknik (EYT)
Abstract: The current multidisciplinary interests on human intestinal microbiomes have stimulated large scale research initiatives, involving collection and processing of up to thousands of fecal samples within a single study. Hence, there is a need for high throughput protocols that are cost-efficient and validated for their performance to ensure that the relative abundance of different bacteria, the main outcome of microbiota studies, is not biased due to technical artefacts originating from sample processing. Infant’s microbiota colonization is one of the central research areas in human microbiome research because of the long-lasting and profound health implications of the pioneering microbes. This experimental study aimed to develop and validate a high throughput fecal sample collection and processing system for microbial DNA extraction operating in 96 well format. This newly developed method was used to extract DNA from 647 fecal samples collected from mother-infant pairs within a clinical study that will study the effect of antenatal antibiotic prophylaxis on infant’s gut microbiota development. A subset of 28 mother-infant pair samples (14 from each antibiotic and non-antibiotic groups) were selected to study the prevalence of a probiotic bacterium, Lactobacillus rhamnosus GG (LGG), among infants and their mothers longitudinally from birth to 3 months by using species-specific PCR amplification method targeting sortase C gene. In addition, the prevalence of L. rhamnosus GG in 3-month-old infants was compared between the above samples and those (n=30) collected in another clinical trial conducted ~10 years earlier. From extensive testing and validation, an efficient high throughput system for fecal sample collection and processing for extraction of microbial DNA in 96 well format was established. Tests were performed to validate the performance of a) fecal sample collection system b) commercial, readymade bead beating tubes for bacterial cell lysis and c) selfmade wash buffers as part of the automatic DNA purification system. Performance was evaluated based on the quality and quantity of the resultant DNA. We show that this new fecal processing system can yield high quality microbial DNA from 96 fecal samples within ~6 hrs. Based on the ratios of dominant gram-positive and gram-negative bacteria evaluated using PCRs and next generation sequencing, the new DNA extraction method resulted into similar microbiota composition than the previously validated manual DNA extraction method. However, the DNA yield per sample was markedly lower due to the lower input volume of the sample. Based on the sortase C gene PCR tests, the prevalence of LGG was similar (~60%) among 3-month-old children in both clinical studies conducted ~10 years apart, although false negatives among the recent samples due to the low amount of DNA cannot be excluded. Following the temporal pattern of colonization, we observed no evidence for the transfer of LGG at the time of birth from the mother to her child, instead the infants became positive for LGG typically between 1-3 weeks after birth. The carriage of LGG seemed to be dependent on their diet. During this project, we found out that the PCR method employed for detection of LGG was not fully specific for this strain, and hence a more specific qPCR assay was developed.

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