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

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.

Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

“…In this study, although post-sequencing analysis revealed comparable performances between the BT and ZB kits across multiple types of respiratory samples, the impact of sample type on other DNA extraction methods requires further investigation. For example, between-kit differences appear to be magnified in gut samples [40, 45] and reduced in respiratory samples [42, 43]. Nevertheless, both BT and ZB kits performed similarly in terms of extracting specific bacteria in the mock community and showed differences in bacterial compositions and diversity metrics between different respiratory sites (Tables 2 and 3), and have therefore validated our previous reports [2, 15–17].…”

“…This normalization appeared to eliminate between-kit differences in the number of 16S rRNA sequences (Fig 3B). Studies evaluating kit effects on bacterial diversity typically focus on the differences observed in the concentration of 16S rRNA copies detected via quantitative PCR without paying much attention to the actual number of 16S rRNA sequences recovered [40, 42]. Furthermore, the contribution of unnormalized DNA concentrations to the observed microbiota profile differences between kits needs to be clarified.…”

Optimizing microbiome sequencing for small intestinal aspirates: validation of novel techniques through the REIMAGINE study

“…Although standards have been set for the assessment of the stool microbiome, these standards have not been assessed for small intestinal fluid assessment. Mucous in general is a viscous fluid that can trap bacteria in its matrix and previous studies performed with sputum samples have shown that treating this viscosity has an impact on the microbial assessment [5,16]. However, until now, no studies have investigated the impact on microbial assessment and DNA recovery in aspirates collected from small bowel.…”

High‐Throughput Metagenomics for Identification of Pathogens in the Clinical Settings

“…The existing sputum liquefaction schemes include the following methods: i) standard N-acetyl-l-cysteine treatment and digestion with 2% NaOH and decontamination; ii) NaOCl liquefaction and sedimentation; iii) chitin solution liquefaction; iv) the use of sputum and an equal volume of phosphate buffer containing 1 g L −1 protease K to liquefy sputum; and v) classical dithiothreitol (DTT) liquefaction, in which a chitin solution homogenizes the mucus sputum more quickly than the N-acetyl-l-cysteine and NaOCl methods. [47][48][49] With the advancement of automated processing, a portable, low-power preprocessing device can perform all of the required steps for sputum preprocessing, including liquefaction, homogenization, dissolution, and inactivation. [50]…”