Background: Healthcare-associated pneumonia (HCAP) is a heterogeneous disease. Previous studies reported that identifying risk factors for HCAP may lead to more accurate predictions of drug-resistant pathogens. We examined new nursing-home- and hospital-associated infections (NHAI) by revising existing HCAP risk factors. Our aim was to determine whether respiratory microbiota profiles are related to newly defined risk factors for NHAI in critically ill patients on mechanical ventilation.Methods: The NHAI group comprised nursing home residents with a poor functional status, or recent (past 90 days) hospitalization or recent (past 180 days) antibiotic therapy. Endotracheal aspirates were prospectively collected from patients in the intensive care unit (ICU) of a university hospital. The 180 endotracheal aspirates from 60 mechanically ventilated ICU patients (NHAI group, n = 24; non-NHAI group, n = 36) were obtained on days 1, 3 and 7. The bacterial community profiles of the endotracheal tube aspirates (ETAs) were explored by 16S rRNA gene sequencing. A phylogenetic-tree-based microbiome association test (TMAT), generalized linear mixed models (GLMMs), the Wilcoxon test and the reference frame method were used to analyze the association between microbiome abundance (number of operational taxonomic units [OTUs]) and disease phenotype.Results: The microbiome analysis revealed significantly lower α-diversity in the NHAI group than in the non-NHAI group. In the analysis of β-diversity, the structure of the microbiome also differed significantly between the two groups (weighted UniFrac distance, Adonis, P <0.001). The abundance of Corynebacterium was significantly higher, and the relative abundances of Granulicatella, Staphylococcus, Streptococcus and Veillonella were significantly lower, in the NHAI than in the non-NHAI group. Conclusions: The microbiota signature of the ETAs distinguished between patients with and without risk factors for NHAI. Thus, the lung microbiome may serve as a biomarker and therapeutic target for intubated patients exposed to nursing home and hospital environments.