Background Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time. Methods Twenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time. Results The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits. Conclusions The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

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Background Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with cystic fibrosis (CF). Herein, we describe our experience managing a young woman suffering from CF with XDR P. aeruginosa who underwent lung transplantation. We highlight the contemporary difficulties reconciling the clinical, microbiological, and genetic information. Methods Mechanism-based-susceptibility disk diffusion synergy testing with double and triple antibiotic combinations aided in choosing tailored antimicrobial combinations to control the infection in the pre-transplant period, create an effective perioperative prophylaxis regimen, and manage recurrent infections in the post-transplant period. Thirty-six sequential XDR and PDR P. aeruginosa isolates obtained from the patient within a 17-month period, before and after a double-lung transplant were analyzed by whole genome sequencing (WGS) and RNAseq in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time Results Our phylogenetic reconstruction demonstrates that these isolates represent a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggests that a group of closely related strains was present in the patient prior to transplantation and continued to evolve throughout the course of treatment regardless of antibiotic usage.Our findings challenge antimicrobial stewardship programs that assist with the selection and duration of antibiotic regimens in critically ill and immunocompromised patients based on single-isolate laboratory-derived resistant profiles. We propose that an approach sampling the population of pathogens present in a clinical sample instead of single colonies be applied instead when dealing with XDR P. aeruginosa, especially in patients with CF. Conclusion In complex cases such as this, real-time combination testing and genomic/transcriptomic data could lead to the application of true “precision medicine” by helping clinicians choose the combination antimicrobial therapy most likely to be successful against a population of MDR pathogens present. Disclosures Federico Perez, MD, MS, Accelerate (Research Grant or Support)Merck (Research Grant or Support)Pfizer (Research Grant or Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Background Meropenem-vaborbactam (MV) is the first carbapenem/β-lactamase inhibitor combination developed to restore meropenem susceptibility against KPC-producing carbapenem-resistant Enterobacterales(CRE). Vaborbactam (VAB) potently inhibits Ambler class A and C β-lactamases by reversible covalent binding of boronate to serine side chains of β-lactamases. Resistance to MV in non-metallo-β-lactamase (MBL) producing Klebsiella pneumoniae (KP) isolates has been described but remains rare. We sought to identify the major molecular mechanisms associated with MV resistance in KPC-producing KP (KPC-KP) isolates. Methods Clinical isolates with elevated MV MICs were identified by the consult service. Additional clinical isolates with mutations in ompK35 or ompK36 genes were selected from a historic database. Isolates with MBL or OXA-48-like genes were excluded. Controls were comprised of MV susceptible KPC-KP isolates. MICs determination was done using Sensititre automated broth microdilution (BMD) according to CLSI. VAB and avibactam concentrations were held at 8 µg/ml and 4 µg/ml, respectively. Whole genome sequencing (WGS) was performed on all isolates. Genome libraries were prepared using Illumina Nextera XT and sequencing was performed on MiSeq and MinION. Results A total 119 KPC-KP isolates were included in the study. All isolates were resistant to meropenem. Twenty-one KPC-KP with elevated MV MICs were identified. All MV resistant isolates harbored mutations in ompK36 genes. Glycine/aspartate (GD 134-135) insertion, premature stop codon in ompK36 genes, and concomitantly elevated blaKPC copy number were predominant among MV resistant isolates. No insertion elements in ompK36 gene promoter region were found. Two MV resistant isolates exhibited unique mutations in blaKPC and envZ genes. See table for WGS and MIC results. Table 1.Whole genome sequencing and MICs of MV resistant KPC-KP isolatesα: Truncated at nodes 14 and 76, partial genotype consistent with blaSHV-12WT: Wild type*: Premature stop codonGD: Duplication of Glycine (G134) and Aspartate (D135)FS: Frameshift mutationins: insertionMEM: meropenemMVB: meropenem-vaborbactamCZA: ceftazidime-avibactamCFD: cefiderocolN/A: not availableTable 2.Whole genome sequencing and MICs of MV susceptible KPC-KP isolates Conclusion MV resistant KPC-KP isolates were reliably analyzed using WGS to reveal the contribution of omp gene mutations and blaKPC copy number to this phenotype. Elevated MV MICs were additionally recognized among clinical isolates from a historic database preceding MV availability. In the absence of MBL production, caution remains warranted with the use of MV empirically against KPC-KP due to non-β-lactamase mediated resistance mechanisms. Disclosures Daniel D. Rhoads, M.D. PhD, Luminex: Advisor/Consultant|Talis Biomedical: Advisor/Consultant|Thermo Fisher: Advisor/Consultant Federico Perez, M.D., Accelerate: Grant/Research Support|Merck: Grant/Research Support|Pfizer: Grant/Research Support Robert A. Bonomo, MD, NIH VA: Grant/Research Support|VenatoRx Merck Wockhardt Cystic Fibrosis Foundation: Grant/Research Support.