Culture-independent techniques have revealed the composition of a stable microbiota within the distal airways; and although normality criteria have not yet been established, atypical compositions linked to certain respiratory diseases have been detected.1 Dysbiosis is typically detected surrounding tumours tissues, and this trait has been poorly explored in the respiratory airway tissues,2,3 mostly using surgical samples.4–8 An understanding of the microbiota is necessary to decipher its possible causal role in cancer, and also to elucidate the prognosis and response to immunomodulatory therapies, because microorganisms and/or their metabolites shape the local microenvironment, influence the immune response, and impact the final battle against cancer.9 Finally, the presence and/or abundance of specific bacteria could be used as a biomarker, as occurs with Streptococcus gallolyticus sbsp. gallolyticus in the colorectal cancer.10

The aims of the present study were to define the bacterial and fungal microbiota of central lung cancer, in relation to the corresponding contralateral bronchus and compared with controls without cancer history, also defining the core of those microbiotas; and to correlate the pulmonary microbiota in lung cancer with the salivary and faecal compartments.

The role of microbiota in carcinogenic processes has not yet been elucidated and will probably be different for each tumour and localization. Recent data clearly indicate that the microbiota contributes to the prognosis of cancer and determines the response to treatments, particularly responses to the new immunomodulation therapies.9,15 Criteria for the normal composition of lung microbiota have not yet been established, but the available data indicate that their composition in cancer patients differs considerably from that of healthy individuals.2–8 Sampling in the respiratory system requires invasive methods, and here we have characterized the respiratory microbiota surrounding central lung cancer by direct sampling of the tumour tissue by bronchoscopy. Our results reinforce the previously known particularities of the lung microbiota, which considerably differ from oral and stool microbial communities.16 Our results also allowed us to rule out significant contamination with the upper microbiota during the bronchoscopy, as other authors had previously suggested.17–19 Moreover, we considered strict inclusion criteria to avoid the possible bias of antibiotic or corticosteroid therapy, and the bacterial exchange between the anatomically separated niches such as saliva and faeces. Finally, the mycobiome composition of central lung cancer was studied.

Lung microbiota has been described based on health status or a lung cancer diagnosis in sputum,20 bronchoalveolar lavage,5 protected specimen brushing,6 cytological brushing,21 and surgical tissue.7,8,22 Our major contribution is the depiction of the microbiome surrounding central cancer via direct sampling, but our results are not necessarily applicable to the microbiota of the distal airway. Curiously, higher diversity indexes have been detected in bronchi than in faecal or oral compartments, contrarily to the biomass decreases from upper to lower tract described in healthy people.19

We found significantly higher alpha diversity values in cancer than in the control group, whereas other authors have published analogous8 and opposing results.5,6,22 Moreover, advanced cancer stages16 and reduced recurrence-free survival and disease-free survival22 have been associated with higher values of alpha diversity. In that sense, 80% of our patients were at tumour stage III or IV, and their mean survival was only of 198 days in the follow up. Furthermore, other factors such as environmental exposure, residence in high-population density areas,4,16 and pack-years of tobacco smoking, can increase the biodiversity of the lung microbiota, whereas chronic bronchitis reduces it.16 All our patients had been smokers, while most of the controls (56%) had not (Table 1).

In terms of beta diversity, cancer affected and contralateral bronchi had very similar compositions, probably reflecting the environmental influence which is not restricted to the cancer area.2,6,21,22 Although the limited size of our sample prevents us from reaching solid conclusions, no differences were detected in the composition of the bronchial microbiota as a function of the histological variants of the cancer. The abundance of Firmicutes to the clear detriment of Proteobacteria was the most noticeable result in our patients, and this result was consistent in all samples. Proteobacteria dominance in health lung microbiota, especially Pseudomonas, has been also previously corroborated.5,6,23 Higher concentrations of Streptococcus, Blautia, Akkermansia, and Rothia were observed in patients, but Streptococcus abundance was consistently the major marker linked to lung cancer. This fact has been previously reported in saliva,5 sputum,20,24 bronchoalveolar lavage,5 lung tissue,7 and protected specimen brushing.6,21 The exhaustive phylogenetic analysis of ASVs allows us to suggest that the streptococcal variants present in lung tissue are similar to those found in saliva or faeces. New studies including Streptococcus cultures and molecular characterization of the species are needed to decipher whether oral lineages are different from those found in the lungs or faeces and thus establish whether there are any markers truly associated with lung cancer, as occurs with Streptococcus gallolyticus subsp. gallolyticus and colorectal carcinoma.10,25

There is increasing evidence of a link between Streptococcus and lung cancer. Recently, Tsay et al.21 detected Streptococcus and Veillonella enrichment in the lower airways with ERK and PI3K pathways upregulation – an early event that contributes to cell proliferation, survival and tissue invasion – combining microbiome and transcriptomic signatures. The major question that a remains to be answered is whether the abundance of streptococci is a cause or consequence of the tumour process.26Streptococcus is a natural inhabitant of the oral cavity, which is connected to the lower respiratory tract by the larynx and trachea, and the oral/lung bacterial exchange could occur via microaspirations.6,21,23

Microaspiration events are common, but their frequency is significantly increased in chronic inflammatory airway diseases,23 inducing Th17 lymphocytes, as well as expression of inflammatory cytokines (as IL-1α, IL-1β and IL-17). IL-23/IL-17 axis alteration is well known in the pathogenesis of both autoimmune diseases and tumours, and Streptococcus mitis facilitate the cancer development and expansion by induction of IL-1β, IL-6, IL-10 and IL-23 transcription, Th17 activation, and an increased immune checkpoint PD-L1 expression.27 Lung resident γδ T cells with protective roles or pro-tumorigenic functions in cancer have been recently discovered, and local lung microbiota, including Streptococcus, can provoke inflammation and tumour cell proliferation via lung resident γδ T cells activation.28 Our results demonstrated a global streptococcal enrichment in patients with cancer that affected more than just the respiratory tract, supporting the idea that microorganisms can orchestrate the balance between tumour-promoted inflammation and anti-tumour immunity depending on the specific microenvironment.

Streptococcal relative abundance in bronchial biopsies was a good predictor of lung cancer, but unfortunately was not reproducible in saliva. ROC curves suggested the contralateral bronchi as the best sample (90.9% sensitivity and 83.3% specificity; AUC=0.897). Other authors found similar results in protected specimen brushing samples (87.5% of sensitivity and 55.6% specificity, AUC=0.693).6 The proportional abundance of Streptococcus should be validated in the early stages of lung cancer with subsequent follow-up to corroborate their potential use as biomarker. Along these lines, the intestinal enrichment of Streptococcus should be exhaustively explored to identify lung cancer markers in faeces.

The intestinal and the respiratory ecosystems harbour a diverse and abundant microbiota, but some particularities distinguish both ecosystems. Food intake favours a higher rate of microbial reproduction increasing the total mass that is significantly reduced after defecation. On the contrary, nutritional sources for bacteria in the airway are limited to mucus and cellular debris, while clearance is carried out by the ciliary system and the immune system, particularly by macrophages. Lung microbiota core, which is composed by those taxa that are present in >95% of individuals, had not yet been defined. We have implemented new computational strategies to define the lung microbiota core, which had not previously been defined. Our main results are the elevated alpha-diversity of the bronchial microbiota in comparison with saliva or faeces, and the dominance of Pseudomonas in controls. Presence of this particular genus is linked to cystic fibrosis, being the major pathogen that decreases the respiratory functionality within a pathogenic colonization in those patients. However, the lack of respiratory symptoms reduces the potential pathogenic role of Pseudomonas in healthy individuals, although more studies are needed in that line.

The mycobiome results were consistent with those obtained for bacteria. The fungal community was slightly richer and more diverse in patients than in controls, although the contralateral bronchus was more similar to controls than to the affected counterpart. The mycobiome of saliva and the affected bronchus from patients matched perfectly, but differed in controls, again suggesting that in patients with cancer the bronchial microbiota is the result of a continuous exchange with that of the oral niche. In terms of taxonomy, affected bronchi from patients had an enrichment of the Basidiomycota phylum with higher populations of Malassezia genus, whereas the enriched taxon in healthy individuals was the Ascomycota phylum and the genera Candida and Saccharomyces, as previously described.29 Although the public databases are increasing exponentially, it is important to note that a major limitation to describing the mycobiome is the lack of available taxonomic records. As far as we know, this is the first description of the lung mycobiome.

The main limitation of our work is that we cannot estimate the contribution effect of lung cancer factors on the bronchial microbiota, mainly tobacco (all patients had been smokers but only the half of the controls were), and COPD (12/25 patients). However, it has not been established yet if tobacco has a significant influence on lung microbiota composition, and some important studies have shown contradictory results.16,30,31 Whereas severe COPD has been linked with significant alterations in the lung microbiota composition,32,33 mild and moderate COPD (92% of our COPD patients) has been associated with Streptococcus enrichment.33 This finding might explain the association of mild/moderate COPD and lung cancer,34 although further studies are needed to confirm this association.

Additional limitations are the small number of patients, all of them from the same hospital, and the lack of other -omic analyses based on genetic expression. On the other hand, our strengths include performing the first study of microbiota combined with mycobiome of bronchial tissue obtained directly from tumour and contralateral bronchi (not adjacent to a resected tumour), as well as performing analysis of the connected ecosystems including saliva and faeces.

In summary, patients with central lung cancer have a significantly different bronchial microbiota from controls, not restricted to tumour-involved tissue, and probably conditioned by continuous microaspiration events from the oral cavity, more than by the carcinogenic process. Lung cancer was associated with a considerable enrichment of Streptococcus and we propose that this feature could be used for the screening, diagnosis of this pathology. Lung mycobiota differ considerably in control individuals, and there are dissimilarities in patients between the affected and the contralateral bronchi. An innovative bioinformatics strategy used in this study has allowed us to define healthy individuals’ the bronchial core microbiota, which is dominated by a non-pathogenic colonization of Pseudomonas.

The Ethics Committee “CEIC Aragón” approved this project in 2016 with the reference 15/2016, and all participants signed the informed consent after receiving all the information from the clinical researchers.

SB conceived the study. JJV, ALF, EM, and AR contributed to the patients’ selection and sample collection. JMP, JG carried out the computational analysis for core microbiota definition. MPA and RdC determined the microbiota composition. AR, PG, JMP, JG, and SB participated in the analysis and interpretation of data. SB, RdC, and MPA wrote and reviewed the manuscript, and finally all authors read and approved the final version.

This work was partially supported by the project PI17/00115 which recipient is RdC. MPA was supported by the Programa Operativo de Empleo Juvenil, co-financed by the European Social Fund Investing in your future (ESF) and ERDF (PEJD-2018-PRE/BMD-8237), and by a Rio Hortega contract (CM19/00069) from the Instituto de Salud Carlos III.

RdC is the recipient of a Vertex grant IIS-2017-106179 for cystic fibrosis research. The remaining authors declare that they have no competing interest.