How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis.

Leonardo Terranova,Paola Marchisio,Elisa Franceschi,Francesco Blasi,Annamaria Bevivino,Camilla Tafuro,James Chalmers,Samantha Sottotetti,Stefano Aliberti,Antonio Teri,Andrea Gramegna,Lisa Cariani,Martina Contarini,Luca Ruggiero,Martina Oriano

doi:10.3390/ijms19103256

Abstract

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

Highlights

The study of the human microbiome has emerged as an important field in translational research over the past decade, and promising applications of this technique in clinical practice have been suggested [1]
A total of 32 adult patients with cystic fibrosis (CF) and bronchiectasis were enrolled in this study, and 77 sputum samples were collected for the three experiments
We evaluated the use of DTT through 16s rRNA real-time PCR in terms of (1) median levels, in order to understand if the presence of DTT was increasing bacterial DNA recovery; and (2) the coefficient of variation between replicates, in order to understand if the presence of DTT was able to better homogenize sputum and increase data reproducibility (Figure 1)

Summary

Introduction

The study of the human microbiome has emerged as an important field in translational research over the past decade, and promising applications of this technique in clinical practice have been suggested [1]. Multiple studies of the human-associated microbiota have suggested a fundamental role of microbes, and predominantly bacteria, in maintaining health and a role for changes in lung microbiota in contributing to diseases such as chronic obstructive pulmonary disease (COPD), bronchiectasis and cystic fibrosis (CF) [2,3,4,5,6,7]. Potentially less representative of the deep lung as compared with bronchoalveolar lavage (BAL), sputum could represent the preferred non-invasive diagnostic technique to be used in clinical practice for microbiota analysis. Some bacteria require high levels of mechanical disruption or enzymatic disruption in order to achieve efficient lysis, and these include important pathogens in bronchiectasis and cystic fibrosis such as Staphylococcus aureus. [8,9]

Methods

Results

Conclusion