Phylogenetic and spatio-temporal analysis of 253 clinical ST-621 P. aeruginosa isolates.

(A) SNP-based, core genome phylogeny. Outbreak subclones are colored with red (SC1) or blue (SC2) branches. From inner- to outermost, datasets show: the year, the floor, the building, and the source of isolation. Patients with prolonged carriage (>1 year) are indicated. (B) Timeline of isolate collection. Each line indicates a single patient. Isolates are shown as dots colored by the floor the patient was on. Patients with prolonged carriage are highlighted. (C) Mock floor plan of the Main (light blue) and Tower (dark blue) Buildings of Facility A. Wards and floors are numbered. For each floor, a pie chart indicates the proportion of isolates collected from urine, respiratory, or other cultures. Within each ward, isolates are colored by year of collection with a red outline indicating primary isolates. Symbols indicate the presumed origin of infection. (D) Bar charts showing for each year the number of: all P. aeruginosa primary clinical isolates collected at Facility A (bottom), the subset of genome-sequenced primary isolates (middle), and the subset of identified ST-621 isolates (top). Bars are color-coded by the isolation source. (E) Distribution of SNP distances for all inter-patient isolates or subsets collected in the same month, the same ward, or both. Dotted lines indicate the local maxima.

Dated phylogeny of 253 ST-621 outbreak isolates.

Time-stamped phylogeny. Error bars for annotated nodes are shown as gray boxes. Years are indicated on the left, along with the opening of the Main Building (T1), and the Tower Building extension (T2). The MRCA of SC1 and SC2 are indicated in black text above the node. The MRCA of isolates from patients with prolonged carriage are shown in red, indicated by their patient ID. The predicted emergence of select variants are shown in blue. Data showing the floor and source isolates were collected from is shown, as in Figure 1. Select datasets are shown including the antibiotic susceptibility testing results for cefepime (cephalosporin) and imipenem (carbapenem).

Identification of environmental reservoirs of the ST-621 outbreak clone in sink drains.

(A) Photos depict a swab from a sink drain in a patient room and the typical sink design (shallow basins and gooseneck faucets) in facility A. (B) Minimum Spanning Tree of 326 Facility A ST-621 P. aeruginosa isolates, including contemporary isolates collected in 2021 and 2022 from clinical (n= 56) and environmental (n= 17) sources. Isolates from wards #1, 6 and 20 where environmental contamination was identified are colored. Environmental isolates are indicated with the letter E.

Antibiotic resistance and poly-variant sites of 253 outbreak isolates.

(A) Bar charts showing the proportion of clinical isolates resistant to tobramycin, cefepime, imipenem, and ciprofloxacin each year from 2011-2020. (B) Chart of mutations (excluding synonymous) in resistance-associated genes. Each block indicates one distinct mutation observed within the corresponding gene. Blocks are colored by the number of isolates sharing this exact mutation. Blocks are outlined to indicate whether the mutation is found within SC1, SC2, or isolates from both subclones. Mutations causing a predicted loss-of-function (LOF) are indicated with a star. (C) Pruned SNP-based phylogeny showing the acquisition of colistin resistance. Patient ID, day of collection (since the first outbreak isolate), and available colistin MIC are shown on the right. The MRCA of the PhoQ E77fs mutation is indicated with a triangle.

Identification of pathoadaptive mutations.

(A) Distribution of COG functional categories for all genes (Ref) compared to the subset of genes (Sel) with 2 or more distinct mutated sites (excluding synonymous) in outbreak isolates. Significant enrichment in cell wall biogenesis and signal transduction categories are indicated. A positional chart of mutations across the entire chromosome of closed reference genome for isolate 4605 is provided. The number of unique mutation sites within each gene is indicated (top) as well as the number of outbreak isolates which carried each mutation (bottom). Genes with 3 or more distinct mutated sites are labeled and genes involved in cell wall biogenesis and signal transduction genes are color coded. (B) Accumulation of mutations (excluding synonymous) in genes and pathways associated with P. aeruginosa pathogenesis. Each block indicates one distinct mutation observed within a gene part of the indicated functional group (data available in Table S8). Blocks are colored by the number of isolates sharing this exact mutation. Blocks are outlined to indicate whether the mutation is found within SC1, SC2, or isolates from both subclones. Mutations causing a predicted loss-of-function (LOF) are indicated with a star.

Minimum Spanning Trees of Pseudomonas aeruginosa cgMLST.

Isolates are color coded by facility, showing the 10 most prominent. (A) Includes all 5,129 Pseudomonas aeruginosa in the MRSN’s collection from 2011-2020. Clusters of isolates belonging to ST-235, -244, and -621 are indicated. (B) Includes all ST-621 isolates from 2011-2020. Genetic distances higher than 23 allelic differences are indicated.