1246. Clinical isolates of Pseudomonas aeruginosa Harbor Preexisting Changes in TonB-dependent Receptors Associated with Decreased Susceptibility to Cefiderocol

Abstract Background Cefiderocol (FDC) is a novel siderophore cephalosporin that retains activity against MDR gram-negative bacteria. In P. aeruginosa (PA), FDC utilizes TonB-dependent receptors (TBDR) PirA, PiuA, or PiuD to enter the periplasmic space. We recently reported a clinical PA isolate that developed elevated MICs to FDC associated with mutations in genes encoding TBDRs in the absence of prior exposure to FDC. In this study, we investigated the frequency of TBDR mutations not associated with cefiderocol exposure among clinical stains of PA recovered from 1999 to 2018 in a large hospital system in Houston, TX. Methods A total of 212 clinical isolates of PA were screened for mutations in TBDR pathways (pirA/pirRS and piuA/D) via whole genome sequencing. Strains with gene mutations predicted to significantly alter protein function (insertion, deletion, or frameshift) were selected for phenotypic characterization. PA PA01 and 4 clinical PA strains lacking changes in the TBDR genes served as controls. FDC susceptibility testing was performed on Mueller-Hinton agar by Kirby-Bauer disc diffusion (DD). Diameters were measured at 18 h and 48 h to assess for the emergence of colonies inside the zone of inhibition. Breakthrough colonies were isolated on cetrimide agar, and DD studies were performed to determine FDC susceptibility. Results 6.1% of isolates (13/212) had preexisting mutations in the TBDR genes, including indels in piuA (n=2) and pirR (n=2), and a frameshift mutation resulting in premature stop codon in pirR (n=9). DD showed that isolates with predicted changes in TBDRs had a significantly smaller diameter of inhibition, as compared to controls (Fig 1). Of the PiuA or PirR mutants, 3 of 13 demonstrated breakthrough colonies (Fig 2); while none of the control specimens showed breakthrough colonies. Subcultures of isolated breakthrough colonies yielded more homogenous populations of PA with relatively lower DDs than the original strain (Fig 2). Figure 1 Cefiderocol disk diffusion diameters of P. aeruginosa isolates at (a) 18 hours growth and (b) 48 hours growth. P-values for student’s two-tailed t-test are given. Dotted lines represent cutoffs for intermediate and resistant per Clinical Laboratories and Standards Institute (CLSI) guidelines. ns, non-significant; TBDR, TonB dependent receptor. Figure 2 Disk Diameter resistance phenotypes suggestive of heteroresistance among P. aeruginosa strains containing TBDR mutations (row 1) and their subsequent breakthrough colony subculture resistance phenotypes (row 2) at 48 hour time points. Conclusion Mutations in genes encoding TBDR are present in clinical isolates of PA that predate the approval of FDC and are associated with the emergence of reduced susceptibility to FDC. Disclosures Truc T. Tran, PharmD, Merck (Grant/Research Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support


Conclusion.
These in vitro data suggest that CAZ-AVI can be an effective treatment option for infections caused by MDR Enterobacterales and P. aeruginosa collected in Latin America.
Disclosures Background. Ceftaroline fosamil, the prodrug of ceftaroline, is a parenteral cephem approved for the treatment of patients with skin and skin structure infections (SSSIs) caused by Staphylococcus aureus (both methicillin-susceptible [MSSA] and methicillin-resistant [MRSA] isolates), β-hemolytic streptococci (Streptococcus pyogenes, S. agalactiae, S. dysgalactiae), and select species of Enterobacterales (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca). The current study is part of the ATLAS (Antimicrobial Testing Leadership and Surveillance) program and evaluated the current activities of ceftaroline and comparator agents against commonly encountered bacterial isolates associated with SSSIs.
Methods. From 2012 to 2019 the ATLAS program received 124,694 bacterial isolates that had been cultured by 493 clinical laboratories in 71 countries from samples of patients diagnosed with SSSIs. All isolates were transported to IHMA, (Schaumburg, IL, USA) where their identities were confirmed using MALDI-TOF mass spectrometry and antimicrobial susceptibility testing performed following standardized CLSI broth microdilution methodology (M07). Percent susceptibilities were determined using 2021 CLSI MIC breakpoints. Phenotypic extended-spectrum β-lactamase (ESBL) screening and confirmatory testing were performed using the CLSI M100 method.
Results Table   Conclusion. Ceftaroline continues to demonstrate potent in vitro activity against clinically relevant pathogens associated with SSSIs. Disclosures

Clinical isolates of Pseudomonas aeruginosa Harbor Preexisting Changes in TonB-dependent Receptors Associated with Decreased Susceptibility to Cefiderocol
Stephanie L. Egge, MD 1 ; Shelby Simar, MPH 2 ; An Q. Dinh, BS 3 ; Blake Hanson, PhD 2 ; Truc T. Tran, PharmD 3 ; Cesar A. Arias Background. Cefiderocol (FDC) is a novel siderophore cephalosporin that retains activity against MDR gram-negative bacteria. In P. aeruginosa (PA), FDC utilizes TonB-dependent receptors (TBDR) PirA, PiuA, or PiuD to enter the periplasmic space. We recently reported a clinical PA isolate that developed elevated MICs to FDC associated with mutations in genes encoding TBDRs in the absence of prior exposure to FDC. In this study, we investigated the frequency of TBDR mutations not associated with cefiderocol exposure among clinical stains of PA recovered from 1999 to 2018 in a large hospital system in Houston, TX.
Methods. A total of 212 clinical isolates of PA were screened for mutations in TBDR pathways (pirA/pirRS and piuA/D) via whole genome sequencing. Strains with gene mutations predicted to significantly alter protein function (insertion, deletion, or frameshift) were selected for phenotypic characterization. PA PA01 and 4 clinical PA strains lacking changes in the TBDR genes served as controls. FDC susceptibility testing was performed on Mueller-Hinton agar by Kirby-Bauer disc diffusion (DD). Diameters were measured at 18 h and 48 h to assess for the emergence of colonies inside the zone of inhibition. Breakthrough colonies were isolated on cetrimide agar, and DD studies were performed to determine FDC susceptibility.
Results. 6.1% of isolates (13/212) had preexisting mutations in the TBDR genes, including indels in piuA (n=2) and pirR (n=2), and a frameshift mutation resulting in premature stop codon in pirR (n=9). DD showed that isolates with predicted changes in TBDRs had a significantly smaller diameter of inhibition, as compared to controls (Fig 1). Of the PiuA or PirR mutants, 3 of 13 demonstrated breakthrough colonies ( Fig  2); while none of the control specimens showed breakthrough colonies. Subcultures of isolated breakthrough colonies yielded more homogenous populations of PA with relatively lower DDs than the original strain (Fig 2).  Background. The molecular epidemiology of carbapenem-resistant Klebsiella species is not well investigated in Qatar. The objective of this work was to characterize the genetic context of carbapenemase-producing Klebsiella isolates recovered from clinical specimens.
Methods. Klebsiella isolates (n=100) were collected at 7 tertiary hospitals from 2015-2017. Identification and susceptibility testing were performed using MALDI-TOF MS and BD Phoenix system, respectively. Whole Genome Sequencing was performed on the Illumina NextSeq platform. Phylogenomic analysis, screening of resistance and virulence genes, and comparison of genetic environment of carbapenemase were carried out.