Dynamics of the lung microbiome in intensive care patients with chronic obstructive pulmonary disease and community-acquired pneumonia

Little is known about the composition and clinical implications of lung microbiome in patients with chronic obstructive pulmonary disease (COPD) and community-acquired pneumonia requiring invasive mechanical ventilation and intensive care unit admission. Therefore, this study aimed to explore the longitudinal changes in microbial airway composition and its variations between COPD patients with different weaning outcomes. Fifty-one endotracheal aspirate samples from 21 participants and 5 saline samples were collected as the patient and control group, respectively. Sequence analysis revealed significant increases and upward trends in the relative abundance of the Acinetobacter genus and Acinetobacter baumannii complex species in paired comparisons of sampling points and over time, respectively, in patients with failed weaning (p for trend = 0.012 and 0.012, respectively) but not in those with successful weaning (p for trend = 0.335 and 0.426, respectively). Furthermore, significant changes in the composition of the bacterial community were observed in paired comparisons of sampling points in patients with failed weaning compared with those with successful weaning. The alpha diversity did not differ between the patients with different weaning outcomes. These results further the understanding of longitudinal airway microbiome structure analysis and its clinical implications when managing critically ill patients with and without COPD.

were also recorded. In addition, the modified Glasgow Coma Scale with verbal score as one, 5 previous history of admissions and use of antibiotics and systemic steroids within the 3 months prior to study entry were also recorded. The pneumonia severity index, which classifies patients with CAP into five ordered risk classes, 6,7 chest X-ray findings, initial laboratory findings and ventilator settings upon arrival at the RICU were recorded. Other data collected included Acute Physiology and Chronic Health Evaluation II score, co-morbidities, microbiological testing of endotracheal aspirates at the sampling time points of interest which were evaluated by the central laboratory of the study institute based on standard procedures, 8,9 RICU length of stay, and weaning outcomes. All patient information was anonymized and de-identified prior to analysis.

Supplementary Methods S3-Weaning process and outcomes
During the study period, the same intensivist team worked in the RICU where consistent protocol-driven ventilator weaning was applied and implemented according to the standards of the RICU at the study institute. The weaning process has been previously reported, 10 in which successful weaning was defined as liberation from IMV support on discharge from the RICU. Otherwise, the patients were defined as having failed weaning. 10 (PCRs) were performed in a 25μL volume with 0.2μL AccuPrime Taq DNA Polymerase, high fidelity (Thermo Fisher Scientific), 0.5μM forward and reverse primers, and about 1 ng template DNA. Thermal cycling was performed with an initial denaturation step at 94˚C for 2 min, followed by 30 cycles of 94˚C for 20 sec, 56˚C for 30 sec, and 68˚C for 60 sec, then a final elongation step at 72˚C for 5min.
Equal volumes of 1x loading buffer (containing SYB green) and PCR products were mixed and electrophoresis on 2% agarose gel was performed for detection. Samples with 1 main bright strip between 450-500 bp were chosen for further experiments. Then, the mixed PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN). Quantitative analysis of the DNA from the gel extraction was performed using a Qubit dsDNA HS assay kit (Thermo Fisher Scientific).
The fragments generated had single-stranded, 'sticky' ends. The next step, called endrepair, fills in these sticky ends to create blunt ends, ready for adaptor ligation. Adaptors are then bound to both the 5' and the 3' ends of the library fragments. They are specific to the sequencing platform, but ultimately all serve to enable in-platform clonal amplification, i.e.
Illumina's bridge amplification. The adaptors are designed to bind to the sequence-specific substrate, such as a patterned flow cell, and contain sequences to enable amplification, and can have barcodes for fragment identification. Finally, the library was sequenced on an Illumina Miseq platform, generating 300 bp paired-end reads. The library quality was assessed using the Qubit 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer1000 system.
Sequenced reads were filtered for quality and processed using the latest version of bioinformatics software package "QIIME v1.80". Operational taxonomic units were clustered from chimera-cleaned reads at a 97% identity threshold and assigned taxonomy through the SILVA-based (version 132) reference database.

Supplementary Methods S5 -16S quantitative polymerase chain reaction
Acinetobacter baumannii complex species was quantified using a quantitative polymerase chain reaction using published primers and probes for the endotracheal aspirate samples. Reactions were performed on an ABI ViiA7 Real-time PCR System (Applied Biosystems). The thermal-protocol was as follows: 50℃ for 2 min, 95℃ for 10 min,

Supplementary Methods S6-Statistical analysis
All data were expressed as the median and interquartile range or as a number (percentage).
Comparisons were performed using the Mann-Whitney U test for continuous variables and chi-square test for categorical variables. A volcano plot was generated using SPSS to display taxa with large magnitude changes that were also statistically significant. The trend analysis of relative abundances of genus Acinetobacter, species A. baumannii complex, and alpha diversity over time was analyzed using the Jonckheere-Terpstra test. Alpha diversity of the samples was assessed using the Shannon index, observed species index, and Chao1 index.
Species relative abundance data were analyzed using principal coordinate analysis, which was conducted with R package vegan based on Bray-Curtis distance. Between-group inertia  ＆ Major antibiotics were defined as an antimicrobial that was given for 4 days or more in a week. # Modified GCS score, verbal score as one.
Abbreviations: APACHE II score, Acute Physiology and Chronic Health Evaluation II score; BMI, body mass index; BUN, blood urea nitrogen;