Development of a robust protocol for the characterization of the pulmonary microbiota

Abstract

The lack of methodological standardization diminishes the validity of results obtained and the conclusions drawn when studying the lung microbiota. We report the validation of a complete 16S rRNA gene amplicon sequencing workflow, from patient recruitment to bioinformatics, tailored to the constrains of the pulmonary environment. We minimize the impact of contaminants and establish negative controls to track and account for them at every step. Enzymatic and mechanical homogenization combined to commercially available extraction kits allow for a fast and reliable extraction of bacterial DNA. The DNA extraction kits have a significant impact on the bacterial composition of the controls. The bacterial signatures of extracted cancerous and healthy human tissues from 5 patients are highly distinguishable from methodological controls. Our work expands our understanding of low microbial burdened environments analysis. This article is to be a starting point towards methodological standardization and the implementation of proper sampling procedures in the study of lung microbiota.

Non-small cell lung cancer microbiota characterization: Prevalence of enteric and potentially pathogenic bacteria in cancer tissues

“…Therefore, the tissue samples and the controls share very few OTUs in potentially different proportions and the influence of contaminants on the bacterial composition detected for tissues seems negligible. The removal of the contaminating OTU was still performed as described by Dumont-Leblond et al [22,24]. The results presented here are a reliable description of the pulmonary microbiota.…”

“…The sampling, sample treatment, bacterial DNA extraction, sequencing and bioinformatics analyses were performed as we described Dumont-Leblond et al [22]. Briefly, a whole section of tumors of 5 mm thickness, 8 cm 3 of healthy tissue located 5 cm from tumors in the same pulmonary region, and a methodological control accounting for every step of the protocol were extracted and sequenced for each patient.…”

“…The DNA extraction method is also designed to be completed in a single tube and by the addition of reagent, which allows us to obtain representative no-template control for each pair on healthy and cancerous tissue. The presence of contaminants was accounted for through bioinformatics by removing Operational Taxonomic Unit (OTU) found in controls from the corresponding samples on a relative abundance basis [22,24].…”

“…As described in the previously published method article [22], methodological controls, accounting for every step of the experimental protocol, from sampling to sequencing, were…”

“…This study aimed at deploying a newly validated methodology to identify the bacterial components of the lung microbiota in cancer patients [22]. Tumoral and healthy tissue samples obtained from patients diagnosed with squamous cell carcinoma or adenocarcinoma that underwent lung resection were characterized.…”