Composition and diversity analysis of the lung microbiome in patients with suspected ventilator-associated pneumonia
Authors: Fenn Dominic , Abdel-Aziz Mahmoud I. , van Oort Pouline M. P. , Brinkman Paul , Ahmed Waqar M. , Felton Timothy , Artigas Antonio , Póvoa Pedro , Martin-Loeches Ignacio , Schultz Marcus J. , Dark Paul , Fowler Stephen J. , Bos Lieuwe D. J. ,   The BreathDx Consortium, Ahmed Waqar M. , Raventos Antonio Artigas , Bannard-Smith Jonathan , Bos Lieuwe D. J. , Camprubi Marta , Coelho Luis , Dark Paul , Davie Alan , Diaz Emili , Goma Gemma , Felton Timothy , Fowler Stephen J. , Goodacre Royston , Johnson Craig , Knobel Hugo , Lawal Oluwasola , Leopold Jan-Hendrik , Martin-Loeches Ignacio , Nijsen Tamara M. E. , van Oort Pouline M. P. , Povoa Pedro , Rattray Nicholas J. W. , Rijnders Guus , Schultz Marcus J. , Steenwelle Ruud , Sterk Peter J. , Valles Jordi , Verhoeckx Fred , Vink Anton , Weda Hans , White Iain R. , Winters Tineke , Zakharkina Tetyana 
Published: July 2022

Abstract
Background
Ventilator-associated pneumonia (VAP) is associated with high morbidity and health care costs, yet diagnosis remains a challenge. Analysis of airway microbiota by amplicon sequencing provides a possible solution, as pneumonia is characterised by a disruption of the microbiome. However, studies evaluating the diagnostic capabilities of microbiome analysis are limited, with a lack of alignment on possible biomarkers. Using bronchoalveolar lavage fluid (BALF) from ventilated adult patients suspected of VAP, we aimed to explore how key characteristics of the microbiome differ between patients with positive and negative BALF cultures and whether any differences could have a clinically relevant role.

Methods
BALF from patients suspected of VAP was analysed using 16s rRNA sequencing in order to: (1) differentiate between patients with and without a positive culture; (2) determine if there was any association between microbiome diversity and local inflammatory response; and (3) correctly identify pathogens detected by conventional culture.

Results
Thirty-seven of 90 ICU patients with suspected VAP had positive cultures. Patients with a positive culture had significant microbiome dysbiosis with reduced alpha diversity. However, gross compositional variance was not strongly associated with culture positivity (AUROCC range 0.66–0.71). Patients with a positive culture had a significantly higher relative abundance of pathogenic bacteria compared to those without [0.45 (IQR 0.10–0.84), 0.02 (IQR 0.004–0.09), respectively], and an increased interleukin (IL)-1β was associated with reduced species evenness (rs = − 0.33, p < 0.01) and increased pathogenic bacteria presence (rs = 0.28, p = 0.013). Untargeted 16s rRNA pathogen detection was limited by false positives, while the use of pathogen-specific relative abundance thresholds showed better diagnostic accuracy (AUROCC range 0.89–0.998).

Conclusion
Patients with positive BALF culture had increased dysbiosis and genus dominance. An increased caspase-1-dependent IL-1b expression was associated with a reduced species evenness and increased pathogenic bacterial presence, providing a possible causal link between microbiome dysbiosis and lung injury development in VAP. However, measures of diversity were an unreliable predictor of culture positivity and 16s sequencing used agnostically could not usefully identify pathogens; this could be overcome if pathogen-specific relative abundance thresholds are used.