Poster #2: Sydney Stanley PhD

Whole Genome Characterization of the Carbapenem-Resistant Enterobacterales Plasmidome Within a Large Academic Pediatric Hospital for Molecular Surveillance

Sydney Stanley, PhD1,2 Dayanara I Torres, BS3,4 Erin Theiller, PSM3,4 Stephanie Higgins, MS3,4 Ahmed M Moustafa, PhD3,4,5,6 Robert F. Potter, PhD1,2,3 1.

Carbapenem-resistant Enterobacterales (CRE) are “superbugs” resistant to carbapenems, potent last-resort antibiotics reserved for the treatment of severe invasive multidrug-resistant bacterial infections. Carbapenem resistance (CR) is plasmid-mediated and sequencing enables high-resolution genomic surveillance beyond species and gene detection, but molecular characteristics of CREs from US hospitals and pediatrics are underexplored due to comparatively lower prevalence. However, 12,700 US CRE infections were reported in 2020 and the incidence increased by 18% between 2019 and 2023, so it is imperative to establish the molecular details of US pediatric CREs for proactive genomic surveillance as cases inevitably rise.  Methods We collected all 24 CREs from the Children’s Hospital of Philadelphia between 2019 and 2025, including Citrobacter freundii (n=1), Enterobacter cloacae (n=1), Escherichia coli (n=10), and Klebsiella pneumoniae (n=12). CR was defined by phenotypic antibiotic susceptibility testing and the GeneXpert Carba-R carbapenemase gene detection molecular test. Whole-genome sequencing with Oxford Nanopore Technology and Illumina enabled generation of long- and short-read sequences to accurately resolve strain chromosome from the plasmids. Multilocus sequence type (ST), antibiotic resistance genes (ARGs), plasmid replicon types, and virulence genes were detected with computational genetic tools.  Results The most common sublineages were ST 258 K. pneumoniae (5/12) and E. coli ST 167 (2/10) and ST 617 (2/10). These CRE STs are globally disseminated, but we also identified E. coli ST 58 and ST 357 as novel CRE clones. Across the cohort, we identified 27 copies of carbapenemase genes encoding KPC-1 (n=6), KPC-3 (n=8), NDM-1 (n=3), NDM-5 (n=7), OXA-181 (n=1), OXA-232 (n=1), and OXA-484 (n=1). Three strains encoded two carbapenemases on separate plasmids (KPC-1/KPC-1, KPC-3/KPC-3, and NDM-5/OXA-181). We found 52 plasmids with 16 different replicon types, mostly IncFIB (n=13) and IncFII (n=10). We also identified the rarer Col440II_1 and ColRNAI_1 CRE replicons. For 19 strains, the plasmids encoding carbapenemases also carried at least one other ARG. Three strains have carbapenemases and virulence factors encoded on the same plasmid, including a ST 39 E. coli with a Col156_1 plasmid type expressing  KPC-1 and the enteroinvasive E. coli enterotoxin TieB.  Conclusions Pediatric CRE plasmid types are diverse, linked to various STs, and carry multiple ARGs and virulence genes. This includes globally prevalent CRE STs and plasmids, but also those that are poorly characterized. This suggests there are gaps in current genomic epidemiological understanding and that improved molecular profiling is imperative to tailor surveillance and diagnostics to CREs relevant the US pediatric context.