We profiled the bacterial, fungal, and viral flora of the lower respiratory tract of patients with ARDS due to COVID-19. The microbiota of the lower respiratory tract of patients with ECMO was rich in Pseudomonas among the bacteria, unclassified fungi among the fungi, and human betaherpesvirus 5 (Cytomegalovirus: CMV) among the viruses.
The Proteobacteria phylum is predominant in the lower respiratory tract of patients with ARDS due to COVID-19, and an increase in the abundance of the Pseudomonas genus and Enterobacter has been reported as a characteristic of the lower respiratory tract of patients with pneumonia due to COVID-19, which is consistent with the results of the present study [14, 15]. Notably, patients with ECMO had a higher relative abundances of Pseudomonas and Klebsiella than those without ECMO, whereas patients without ECMO had a higher abundances of Streptococcus and Staphylococcus aureus. Viral infections damage tissues of the respiratory tract pathway, leading to dysbiosis and the promotion of bacterial colony formation [16]. In COVID-19, Pseudomonas aeruginosa has been reported to promote colony formation, and enrichment of P. aeruginosa is associated with poor prognosis [17, 18]. Enrichment of Pseudomonas may have been greater in ECMO patients with severe ARDS, who have more severe lung injury. The oral microbiota of the elderly is rich in staphylococci and streptococci, and an increased relative abundance of Enterobacter in the gut has been reported in patients with severe ARDS [19–21]. Aspiration in patients with ARDS also affects the microbiota of the lower respiratory tract [22, 23]. Thus, there may be an increase in streptococci in patients without ECMO and a more confirmed presence of Enterobacter predominantly in severely ill ECMO patients. Despite the enrichment and severity of highly pathogenic microorganisms in the ECMO patients, clinical outcomes and diversity were not inferior to those of the non-ECMO patients. ECMO replaces oxygenation and carbon dioxide removal that the lungs would normally do and allows for protective ventilation that significantly reduces plateau and driving pressures [24]. This results in a significant reduction in the concentrations of plasma sRAGE, interleukin-6, and monocyte chemotaxis protein-1, thus limiting pulmonary biotrauma caused by mechanical ventilation [25]. ECMO has also been reported to promote recovery of alveolar epithelial function in rat experiments [26]. The fact that changes in lung microbiota did not lead to a decrease in diversity in the ECMO patients may have contributed to the protective effect of ECMO on the lungs.
The fungal flora of ARDS patients has been reported to be enriched with Candida albicans, and C. albicans is a risk factor for death [27, 28]. This was similarly observed in ARDS with COVID-19, in which an increase in unidentified ascomycetes in ARDS patients who were not contaminated with Candida spp. was reported [29]. This is consistent with the results of the present study, which showed patients contaminated with C. albicans from an early stage and an increase in unidentified fungi in the uncontaminated cases. Notably, M. restricta was abundant in the present study. Malassezia restricta is endemic to the skin and intestinal tract [30], and in the intestinal fungal flora, M. restricta and C. albicans are prominent [31]. Similar to the bacterial flora, M. restricta may have been enriched through the gut-lung axis in ARDS patients.
The respiratory virus flora are thought to play an important role in the pathogenesis of respiratory disease by interacting with the immune system [7, 32]. Tobacco mosaic virus is reported to be abundant in COVID-19 and both Anelloviridae and Redondoviridae are abundant in severe cases, and the presence of these viruses is positively correlated with intubation during hospitalization [33, 34]. In the present study, there was marked enrichment of CMV and Human alphaherpesvirus 1 (Herpes simplex virus 1: HSV-1), but not Anelloviridae and Redondoviridae. The patients with ECMO tended to have less Human betaherpesvirus 5 than those without ECMO. Human betaherpesvirus 5 and Human alphaherpesvirus are the most commonly identified viruses from patients on mechanical ventilation, and viral reactivation among ICU patients, especially among the herpes group, significantly changes the virome [35]. It was reported that CMV pulmonary infections, not HSV-1, were associated with increased length of mechanical ventilation and increased ICU length of stay and mortality in ventilated patients [36]. Given the decreasing trend in the relative abundance of CMV with the increasing duration of mechanical ventilation in ECMO patients, the lung rest provided by ECMO may contribute to the suppression of pathogenic viral enrichment. The subjects of the previously reported studies were early in the initiation of mechanical ventilation, and there are no reports on the progression of single-virus enrichment with increased duration of mechanical ventilation.
Limitations
This study has several limitations. First, although we detected a large number of microorganisms using metagenomic sequencing, contamination in the airways and of bronchoscopes should always be considered. We used disposable bronchoscopes to minimize contamination. Second, the present results are based on measurements from a single center and have not been validated in other cohorts. Third, we did not evaluate whether the microorganisms were truly pathogenic or only present in the airways. Further studies using animal models should provide important insights into the pathogenic role of microbiome alterations in patients with ECMO.