Comparison of different conditions for DNA extraction in sputum - a pilot study

Respiratory microbiome is a topic of great interest nowadays in translational research for chronic respiratory diseases [1]. Most of the published experiences on respiratory microbiome enrolled chronic obstructive pulmonary disease (COPD) and bronchiectasis patients, including those with cystic fibrosis, and sputum has been the sample most commonly used by investigators [2‐5]. Sputum collection represents an easy and non-invasive strategy for studies on respiratory microbiome. So far, sequencing of microbial communities from airway samples through meta-omic approaches, mainly based on high-throughput DNA sequencing techniques, has been confined to scientific research. Although the use of respiratory microbiome analysis has not been implemented in clinical practice yet, a possible role of this technique in stratifying patients for disease severity and predicting clinical outcomes could be considered [6].

The choice of an appropriate methodology for evaluating the microbiome in sputum samples is crucial to identify the largest biodiversity as possible and to obtain reliable and comparable results. It is a precocious phase for the development of DNA extraction methods from sputum and there is an extreme heterogeneity of DNA extraction technique in literature. However, in this respect, several challenges could be identified. First, sputum is a substrate with a complex and very difficult-to-manage matrix. The use of different techniques for DNA extraction from sputum might be limited by its nature. In order to facilitate its treatment, a solubilizing agent, such as dithiothreitol (DTT), is usually used [7]. Second, epidemiological data on chronic respiratory infections in bronchiectasis and cystic fibrosis revealed the presence of hard-to-lyse bacteria, such as Staphylococcus aureus, leading to the possible need of an individualized strategy to enhance bacterial lysis, such as the use of enzymes able to specifically target Gram-positive bacteria [8]. Pre-treatments with lysostaphin and lysozyme, which are able to target cell wall peptidoglycan and pentaglycine bridges respectively, have been reported in literature for microbiome analysis [9, 10]. Third, sputum complex and viscous matrix might represent a limitation for broadly used DNA extraction techniques. Among them, chemical coupled with enzymatic lysis is extensively used to treat biological fluids and tissues, while mechanical disruption is usually suggested for soil or feces. Fourth, the bioinformatic approach is not standardized and several pipelines are constantly emerging that can be applied to the analysis of microbiome data generated using next-generation sequencing (NGS) techniques. Finally, largely different methodologies, including steps ranging from wet laboratory practices to in silico data analysis, have been published in literature, limiting the comparability of results across different experiences [2, 5, 11]. It is, thus, evident the importance of assessing the performance of different methods that can be applied to the analysis of microbiome in sputum samples with a special focus on the use of DTT and lysostaphin.

The objective of this pilot study was to compare different conditions, such as the use of DTT as a homogenizing agent, lytic enzymes in order to specifically target Gram-positives and DNA extraction technique in order to isolate bacterial DNA from sputum in adult patients with bronchiectasis, according to different endpoints from total DNA extraction to microbiome analysis.

Five sputum samples were collected from five adult bronchiectasis patients followed at the Bronchiectasis Program of the Fondazione IRCCS Ca′ Granda Ospedale Maggiore Policlinico, Milan, Italy, in January 2018. Subjects signed an informed consent and gave their approval for using samples for the purpose of this study. Aliquots of at least 5 ml of sputum were collected and stored at − 80 °C for all subsequent analyses.

DNA was recovered from all the five sputum samples. A higher quantity of DNA has been extracted using Roche and Zymo kits in comparison to Mobio one; the median values of extracted DNA across the 6 different conditions for Roche, Zymo and Mobio kits were 26.062, 19.750 and 1.233 μg respectively. Detailed median (IQR) values of extracted DNA for all the 18 different conditions are reported in Fig. 1.

Results of the real-time PCR for 16S rRNA gene for each condition (Fig. 2), show that no significant differences across the 18 conditions (range of medians between 20 and 21 Ct).

Real-time PCR for S. aureus has been performed for all samples and was positive for two of them. The addition of the enzymatic digestion step increased Staphylococcus DNA extraction (Fig. 3). A similar pattern can be observed through all the conditions even if no statistical evidence is present.

In terms of alpha diversity, no significant differences have been detected among the 18 evaluated conditions, neither considering the Shannon index (range of median values: 0.492–0.574), nor the Simpson (range of median values: 0.624–0.692). Evaluation made using ENOS from Shannon index (range of median values: 1.6–1.8) and ENOS from Simpson index (range of median values: 2.7–3.3), did not show any appreciable difference (Fig. 4).

Relative abundances at the genus level of identified bacteria in each sputum sample across the 18 evaluated conditions are reported in Fig. 5. Microbiome composition is very different across different samples. Sputum samples presenting few genera have very similar data, while in the presence of a higher number of bacterial genera (Patient 2 and 4) differences emerge when comparing the same condition with and without DTT. In sputum sample 5, where only two genera are present, there is an increase of Staphylococci presence in samples treated with lysostaphin and lysozyme. Relative abundances of the four most abundant bacteria detected in each sputum sample across the 18 evaluated conditions are reported in (Additional file 1: Table A).

The major finding of the present experience is that the 18 different conditions evaluated seem to equally perform in extracting DNA from sputum samples in terms of Real-Time PCR for 16 s rRNA gene, alpha diversity and relative abundances of bacterial genera. However, the DNA extraction yield seems to be higher if Roche and Zymo kits are used in comparison to the Mobio one.

The use of enzymatic techniques seems to allow a higher DNA extraction yield than mechanical ones. A high yield might be preferred for 16 s rRNA sequencing because sputum is a matrix with a high percentage of human DNA. For this reason, in order to prepare libraries with an acceptable bacterial DNA amount and more representative for the microbiota of the sample, the starting amount of DNA loaded into PCR need to be high. In order to overcome this limitation, it is possible to either increase the amount of sputum collected by patients or, in case of a limited amount of sputum sample, select a technique able to obtain a high yield of DNA (such as Roche or Zymo kits).

Both the evaluated pre-treatments seem to play a crucial role. The addition of lytic enzymes is important to enhance S. aureus DNA extraction; however, an increase of lysostaphin concentration over 0.18 mg/mL seems not to increase the performance of the kit to extract DNA. These results, first identified through Real-Time PCR for S. aureus, are confirmed by an increase in Staphylococcus genus identification through sequencing. The addition of DTT has a role in improving data reproducibility. Through sequencing, we saw that the presence of DTT seemed to be able to better homogenize sputum samples, leading to more reproducible results in terms of bacterial genera detected, and at the same time seems not to affect the efficiency of DNA extraction. Relative abundances in sputa presenting a large number of bacterial genera are very similar in conditions with DTT, while they vary greatly but very different when it is absent. In sputum sample 2 there are differences in extraction of Stenotrophomonas genus, greatly improved in absence of DTT, presence of 0.18 mg/ml lysostaphin and treated with Zymo kit. Comparing this result with the same condition, in presence of DTT we might speculate that the difference is given by sampling. DTT enable the release of bacteria entrapped into sputum matrix. For this reason, the repetition of the analysis on the same sample leads to comparable results. If a homogenization step is not present, differences given by sampling can be present.

This study has some limitations. First, different interventions, such as the use of DTT and lysostaphin, have been performed at the same time, while no sequential approach has been used. Although this might help us in evaluating possible synergies between variables, difficulties in data interpretation might be present without a special focus on a single variable. Second, the monocentric design, the low number of samples collected and the fact that no patients other than bronchiectasis have been enrolled might interfere with the generalizability of our results. Third, only three kits have been considered, although others are on the market. Finally, our study did not take into account the possible presence of initial contamination of commercial kits.

None of the 18 evaluated conditions seems to be superior to the other ones in extracting DNA from sputum samples, although a higher amount of extracted DNA could be obtained using enzyme-based commercial kits. Pre-treatments with lysostaphin, lysozyme and DTT seem to be necessary in order to have the most representative microbiome evaluation possible. Further studies will be necessary in order to confirm our data. Moreover, these preliminary data show that neither synergic nor interfering effect is present between variables. Punctual evaluation of variables in an independent way should be needed in order to better address this issue. The hypothesis of evaluating microbiome from the same sputum samples in accordance with the same standard operating procedures across different international centers will be considered in case of multicenter studies on microbiome analysis will be designed in the next future.