Background Klebsiella pneumoniae strains have been divided into two major categories: classical K. pneumoniae, which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulent K. pneumoniae, which cause severe community-acquired and disseminated infections in normal hosts. Both types of infections may lead to bacteremia and are associated with significant morbidity and mortality. The relative burden of these two types of K. pneumoniae among bloodstream isolates within the United States is not well understood. Methods We evaluated consecutive K. pneumoniae isolates cultured from the blood of hospitalized patients at Northwestern Memorial Hospital (NMH) in Chicago, Illinois between April 2015 and April 2017. Bloodstream isolates underwent whole genome sequencing, and sequence types (STs), capsule loci (KLs), virulence genes, and antimicrobial resistance genes were identified in the genomes using the bioinformatic tools Kleborate and Kaptive. Patient demographic, comorbidity, and infection information, as well as the phenotypic antimicrobial resistance of the isolates were extracted from the electronic health record. Candidate hypervirulent isolates were tested in a murine model of pneumonia, and their plasmids were characterized using long-read sequencing. We also extracted STs, KLs, and virulence and antimicrobial resistance genes from the genomes of bloodstream isolates submitted from 33 United States institutions between 2007 and 2021 to the National Center for Biotechnology Information (NCBI) database. Results Consecutive K. pneumoniae bloodstream isolates (n = 104, one per patient) from NMH consisted of 75 distinct STs and 51 unique capsule loci. The majority of these isolates (n = 58, 55.8%) were susceptible to all tested antibiotics except ampicillin, but 17 (16.3%) were multidrug-resistant. A total of 32 (30.8%) of these isolates were STs of known high-risk clones, including ST258 and ST45. In particular, 18 (17.3%) were resistant to ceftriaxone (of which 17 harbored extended-spectrum beta-lactamase genes) and 9 (8.7%) were resistant to meropenem (all of which harbored a carbapenemase genes). Four (3.8%) of the 104 isolates were hypervirulent K. pneumoniae, as evidenced by hypermucoviscous phenotypes, high levels of virulence in a murine model of pneumonia, and the presence of large plasmids similar to characterized hypervirulence plasmids. These isolates were cultured from patients who had not recently traveled to Asia. Two of these hypervirulent isolates belonged to the well characterized ST23 lineage and one to the re-emerging ST66 lineage. Of particular concern, two of these isolates contained plasmids with tra conjugation loci suggesting the potential for transmission. We also analyzed 963 publicly available genomes of K. pneumoniae bloodstream isolates from locations within the United States. Of these, 465 (48.3%) and 760 (78.9%) contained extended-spectrum beta-lactamase genes or carbapenemase genes, respectively, suggesting a bias towards submission of antibiotic-resistant isolates. The known multidrug-resistant high-risk clones ST258 and ST307 were the predominant sequence types. A total of 32 (3.3%) of these isolates contained aerobactin biosynthesis genes and 26 (2.7%) contained at least two genetic features of hvKP strains, suggesting elevated levels of virulence. We identified 6 (0.6%) isolates that were STs associated with hvKP: ST23 (n = 4), ST380 (n = 1), and ST65 (n = 1). Conclusions Examination of consecutive isolates from a single center demonstrated that multidrug-resistant high-risk clones are indeed common, but a small number of hypervirulent K. pneumoniae isolates were also observed in patients with no recent travel history to Asia, suggesting that these isolates are undergoing community spread in the United States. A larger collection of publicly available bloodstream isolate genomes also suggested that hypervirulent K. pneumoniae strains are present but rare in the USA; however, this collection appears to be heavily biased towards highly antibiotic-resistant isolates (and correspondingly away from hypervirulent isolates).

Pseudomonas aeruginosa is frequently resistant to multiple antibiotics, including aminoglycosides. The rates of resistance to aminoglycosides in bloodstream isolates collected over 2 decades at a United States hospital remained constant, suggesting that antibiotic stewardship programs may be effective in countering an increase in resistance.

Background P. aeruginosa is a cause of hospital-acquired and ventilator-associated pneumonia. Hypermutator P. aeruginosa strains have been described in patients with cystic fibrosis and chronic respiratory infections but are rare in patients with acute P. aeruginosa infection. This case describes a hypermutator strain of P. aeruginosa found in a patient with COVID-19-associated acute respiratory distress syndrome (ARDS). Methods Serial respiratory and blood cultures were collected. Short-read sequencing libraries were prepared using the Illumina Nextera XT kit, and whole-genome sequencing was performed using the Illumina NextSeq platform. Long-read sequencing libraries were prepared from unsheared genomic DNA using ligation sequencing kit SQK-LSK109 and sequenced on the Oxford MinION platform. Single nucleotide variants were identified by aligning reads from each isolate to the complete genome of the first available clinical isolate. Hypermutator assays were performed by measuring the mutation frequency rate for rifampin resistance. Antibiotic minimal inhibitory concentrations (MICs) were performed. Growth curves were performed with a starting OD600 of 0.1 with measurements taken every 30 minutes for 24 hours. Results Seventeen respiratory and five blood isolates were obtained throughout 62 days of hospitalization. Fourteen of the 22 isolates exhibited hypermutator phenotypes by rifampin resistance assays, which demonstrated rapid accumulation of mutations. All five bloodstream isolates were hypermutators. MIC testing noted increased resistance to aminoglycosides, fluoroquinolones, and aztreonam in the hypermutator isolates. All bloodstream isolates descended from a single progenitor noted on whole-genome sequencing. Each hypermutator strain contained a mutation in the mismatch repair gene mutL, previously associated with the hypermutator phenotype. Genetic Tree of Patient Isolates The genetic tree highlights hypermutator versus non-hypermutator single nucleotide variants Conclusion This case was notable for multiple isolates of hypermutator P. aeruginosa that persisted over weeks. The patient’s COVID-19 infection and acute respiratory distress syndrome may have facilitated persistence of the P. aeruginosa lineage, allowing a hypermutator lineage to emerge. Disclosures All Authors: No reported disclosures.

Background Cefiderocol (FDC) is a novel antimicrobial agent used for multi-drug resistant Gram-negative pathogens. To date, reports of mutations in β-lactamase and siderophore complex genes have been described and may contribute to FDC resistance. This case describes a New Dehli metallo-β-lactamase (NDM)-producing strain of Enterobacter hormaechei that developed FDC resistance following antibiotic exposure. Methods Serial respiratory and blood cultures were collected from a lung transplant recipient throughout 72 days of hospitalization. Confirmatory susceptibility and combination minimal inhibitory concentration (MIC) testing were performed using broth dilution and E-test assays. Short-read sequencing libraries were prepared using a seqWell plexWell 96 kit, and whole-genome sequencing was performed using the Illumina NovaSeq platform. Reads from the sample genomes were aligned to the chromosome and three plasmid sequences of reference genome ENCL48880. Results Four serial respiratory E. hormaechei isolates and one blood isolate were evaluated. Although initial isolates were susceptible to FDC (MICs 1-2 µg/mL), two respiratory isolates cultured after 41 days of FDC therapy had MICs of 128 µg/mL. The blood isolate remained FDC susceptible despite respiratory resistance. The combination of ceftazidime/avibactam and aztreonam was determined to be active via synergy MIC testing in all isolates, and aztreonam therapeutic drug monitoring confirmed an adequate dosing strategy. Whole-genome sequencing revealed no nonsynonymous single nucleotide variants (SNVs) within the chromosomes but identified a deletion of a large urease island in the resistant isolates. In four of the five isolates, a plasmid (p48880_mcr) was identified and analyzed for possible contributions to FDC resistance. Enterobacter Isolate Assemblies This figure demonstrates genomic assemblies from the five Enterobacter clinical isolates, noting an absence of sequence from ECResp2. Conclusion This case demonstrates development of FDC resistance in E. hormaechei isolates during a 41 day course of FDC therapy. Possible causes of resistance include a large chromosomal deletion and plasmid alleles, demonstrating a potential novel mechanism for FDC resistance. Partnering molecular testing and enhanced antimicrobial stewardship should be encouraged to optimize selection of regimens and durations to prevent resistance to FDC. Disclosures Michael G. Ison, MD MS, GlaxoSmithKlein: Grant/Research Support|Pulmocide: Grant/Research Support|Viracor Eurfins: Advisor/Consultant Nathaniel J. Rhodes, PharmD, MSc, American Academy of Colleges of Pharmacy: Grant/Research Support|Paratek: Grant/Research Support|Third Pole Therapeutics: Advisor/Consultant.