Abstract
In recent years the study of the commensal microbiota is driving a remarkable paradigm shift in our understanding of human physiology. However, intrinsic technical difficulties associated with investigating the Microbiomics of some body niches are hampering the development of new knowledge. This is particularly the case when investigating the functional role played by the human microbiota in modulating the physiology of key organ systems. A major hurdle in investigating specific Microbiome communities is linked to low bacterial density and susceptibility to bias caused by environmental contamination. To prevent such inaccuracies due to background processing noise, harmonized tools for Microbiomic and bioinformatics practices have been recommended globally. The fact that the impact of this undesirable variability is negatively correlated with the DNA concentration in the sample highlights the necessity to improve existing DNA isolation protocols. In this report, we developed and tested a protocol to more efficiently recover bacterial DNA from low volumes of bronchoalveolar lavage fluid obtained from infants and adults. We have compared the efficiency of the described method with that of a commercially available kit for microbiome analysis in body fluids. We show that this new methodological approach performs better in terms of extraction efficiency. As opposed to commercial kits, the DNA extracts obtained with this new protocol were clearly distinguishable from the negative extraction controls in terms of 16S copy number and Microbiome community profiles. Altogether, we described a cost-efficient protocol that can facilitate microbiome research in low-biomass human niches.
Highlights
In recent years, the study of microbial communities colonizing the human body surfaces have revolutionized many fundamental aspects in the areas of medicine and human biology
We present an efficient extraction protocol to recover bacterial DNA from BALF supernatants sampled from cystic fibrosis (CF) infants and chronic obstructive pulmonary disease (COPD) adults
Microbiome research using low biomass samples requires special attention to prevent the introduction of unacceptable variability in the form of environmental contaminants (Salter et al, 2014)
Summary
The study of microbial communities colonizing the human body surfaces have revolutionized many fundamental aspects in the areas of medicine and human biology These communities of microorganisms are considered critical regulators of the normal physiology, and important indicators of pathological processes linked to human disease (Cho and Blaser, 2012; Knight et al, 2017). Since the preconceived assumption of being a sterile environment, the lower airways and the lungs, have been largely neglected in microbiome research This fact has delayed the realization that chronic respiratory conditions are greatly influenced by the local microbiota of the airways, and by communities inhabiting niches farther away such as those from the gut (Marsland et al, 2015; Dickson et al, 2016b; Budden et al, 2017, 2019; Faner et al, 2017). The existence of a truly microbial ecosystem in the lungs is still seen with skepticism, rather than settled residents, it is thought that the lung microbiota may represent a transient population (Dickson et al, 2015)
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