632 - Within-patient genetic relatedness of Pseudomonas aeruginosa strains from multiple body sites in hospitalized children with bloodstream infection

Publication Number: 632.7110

Carl Britto, Boston Children's Hospital, Boston, MA, United States; Jena Blumenthal, Boston Children's Hospital, Boston, MA, United States; Arolyn Conwill, Massachusetts Institute of Technology, Cambridge, MA, United States; Francois Lebreton, Walter Reed Army Institute of Research, SIlver Spring, MD, United States; Ting Luo, Walter Reed Army Institute of Research, Silver Spring, MD, United States; Patrick Mc Gann, Walter Reed Army Institute of Research, Silver Spring, MD, United States; Thomas J. Sandora, Boston Children's Hospital, Boston, MA, United States; Tami Lieberman, Massachusetts Institute of Technology, Cambridge, MA, United States; Gregory P. Priebe, Boston Children's Hospital, Boston, MA, United States

Background: Pseudomonas aeruginosa (Pa) bloodstream infections (BSI) in hospitalized children carry significant morbidity and mortality

Objective: We hypothesized that within-patient genetic variants of Pa at different body sites would reveal Pa genes important for pathogenicity and antibiotic resistance

Design/Methods: We prospectively enrolled hospitalized patients with Pa BSI and a central venous catheter during hospitalization at Boston Children’s Hospital. Aliquots from positive blood culture bottles were diluted and plated on agar, and at least 24 colonies were randomly picked for whole-genome sequencing (WGS). Other body sites (stool, oral, respiratory tract, skin) were cultured 1-3 days after BSI for potential Pa colonization and, if present, 6-24 colonies per body site were randomly picked for WGS. Patient-specific reference genomes were constructed using long-read sequencing using PacBio, and all isolates were sequenced with Illumina (MiSeq/NextSeq). Single nucleotide variant (SNV) calls were made using the WideVariant pipeline. Clinical data were obtained from electronic medical records. Within-patient phylogenetic trees were constructed. Fisher’s exact test was used to compare proportions of patients with all clonal isolates (0 SNV) among those who did and did not have Pa growth from non-blood samples.

Results: We enrolled 9 patients with Pa BSI (median age 2.9 years, IQR 1.25, 16.05 years; 67% female), 3 with ANC < 500 during BSI. Six patients (67%) also grew Pa from other sites (5 perianal, 2 oral, 3 skin, 4 endotracheal tube or tracheal aspirate). We sequenced 557 Pa colonies (264 from blood, 293 from other sites). Clonal colonies were observed in 3 of 3 patients who did not grow Pa from any other body site, while 0 of 6 patients who grew Pa from other body sites had all clonal colonies (P=0.008 by Fisher’s exact test), having 1-10 SNV per patient (median 5.5; IQR 1-10), of which 0-7 were non-synonymous SNV (median 3; IQR 0.75, 4.75). Neutropenia was not associated with clonality. One patient had an antibiotic resistance-associated mutation in the MexD in colonies isolated from the sputum but not from blood, skin, or stool. Another patient had a W277* mutation in OprD (known to confer meropenem resistance and hypervirulence) in blood, stool, and oral colonies. A third patient had a Y62H mutation in the LPS kinase InaA in blood but not stool colonies.

Conclusion(s): In patients with Pa BSI, within-patient phylogenies of Pa show patterns of body-site-specific adaptation and non-synonymous mutations in genes important for virulence and antibiotic resistance.