Repertoire of intensive care unit pneumonia microbiota

Abstract

Despite the considerable number of studies reported to date, the causative agents of pneumonia are not completely identified. We comprehensively applied modern and traditional laboratory diagnostic techniques to identify microbiota in patients who were admitted to or developed pneumonia in intensive care units (ICUs). During a three-year period, we tested the bronchoalveolar lavage (BAL) of patients with ventilator-associated pneumonia, community-acquired pneumonia, non-ventilator ICU pneumonia and aspiration pneumonia, and compared the results with those from patients without pneumonia (controls). Samples were tested by amplification of 16S rDNA, 18S rDNA genes followed by cloning and sequencing and by PCR to target specific pathogens. We also included culture, amoeba co-culture, detection of antibodies to selected agents and urinary antigen tests. Based on molecular testing, we identified a wide repertoire of 160 bacterial species of which 73 have not been previously reported in pneumonia. Moreover, we found 37 putative new bacterial phylotypes with a 16S rDNA gene divergence ≥ 98% from known phylotypes. We also identified 24 fungal species of which 6 have not been previously reported in pneumonia and 7 viruses. Patients can present up to 16 different microorganisms in a single BAL (mean ± SD; 3.77 ± 2.93). Some pathogens considered to be typical for ICU pneumonia such as Pseudomonas aeruginosa and Streptococcus species can be detected as commonly in controls as in pneumonia patients which strikingly highlights the existence of a core pulmonary microbiota. Differences in the microbiota of different forms of pneumonia were documented.

Figure 1. Microbial profiles of positive BAL fluids from patients and controls.

Figure 2. The phylogenetic tree inferred from bacterial 16S rDNA sequences which were identified in all patients using the Neighbor-Joining and the Kimura 2-parameter methods.

Bacteria which were previously identified in pneumonia are shown in black. Bacteria which have not been previously identified in pneumonia and which were identified in this study only in pneumonia patients are shown in red. Bacteria which have not been previously identified in pneumonia patients and which were identified in this study in both pneumonia and control patients are shown in blue, whereas bacteria which have been identified in this study only in control subjects and not in pneumonia patients are shown in green.

Figure 3. A phylogenetic tree inferred from 16S rDNA sequences of novel bacterial phylotypes.

These novel phylotypes exhibited sequence similarities of less than 98% to known bacteria available in the GenBank database, and they were classified in silico using “Classifier” program. Phylotypes are reported according to their genus or by the last possible classification determined by the program. When possible, phylotypes with the same classification are clustered together. The frequency of phylotypes in each cohort is shown on the right.. Bacteroidetes are shown in purple, Firmicutes in red, Proteobacteria in blue, Actinobacteria in yellow, Acidobacteria in orange and Spirochaetes in green. CAP, community-acquired pneumonia; VAP, ventilator-associated pneumonia; NV ICU-P, non-ventilator ICU pneumonia; AP, aspiration pneumonia; and CS, control subjects.

Figure 4. Differences in bacterial microbiota composition between community- acquired pneumonia (CAP), ventilator-associated pneumonia (VAP), non-ventilator ICU pneumonia (NV ICU-P), aspiration pneumonia (AP) and control subjects (CS) cohorts.

Bacteria are shown according to their classes. Distribution of bacterial classes in each cohort is expressed as a percentage. Bacterial classes and their corresponding colours are indicated in the bottom right corner.

Figure 5. Differences in fungal microbiota composition between community-associated pneumonia (CAP), ventilator-associated pneumonia (VAP), non-ventilator ICU pneumonia (NV ICU-P), aspiration pneumonia (AP) and control subjects (CS) cohorts.

Fungi are presented according to their classes. Distribution of fungal classes in each cohort is expressed as a percentage. Fungal classes and their corresponding colours are indicated in the bottom right corner.