Molecular Characterization of Baseline Enterobacterales and Pseudomonas aeruginosa Isolates from a Phase 3 Nosocomial Pneumonia (ASPECT-NP) Clinical Trial

Abstract

We evaluated β-lactam-resistant Enterobacterales species and Pseudomonas aeruginosa baseline lower respiratory tract isolates collected during the ASPECT-NP phase 3 clinical trial evaluating the safety and efficacy of ceftolozane-tazobactam compared with meropenem for treatment of ventilated nosocomial pneumonia in adults. Isolates were subjected to whole genome sequencing, quantitative real-time PCR for quantification of the expression levels of β-lactamase and efflux genes, and Western blot analysis for detection of OprD (P. aeruginosa only). ESBL genes were detected in 168 of 262 Enterobacterales isolates and among these blaCTXM-15 was the most common, detected in 125 isolates. Sixty-one Enterobacterales carried genes encoding carbapenemases while 33 isolates did not carry ESBLs or carbapenemases. Carbapenemase-producing isolates carried mainly NDM and OXA-48 variants, with ceftolozane-tazobactam MIC values ranging from 4 to 128 μg/mL. Most ceftolozane-tazobactam-nonsusceptible Enterobacterales isolates that did not carry carbapenemases were K. pneumoniae that exhibited disrupted OmpK35, specific mutations in OmpK36, and, in some isolates, elevated expression of blaCTXM-15. Among 89 P. aeruginosa isolates, carbapenemases and ESBL-encoding genes were observed among 12 and 22 isolates, respectively. P. aeruginosa isolates without acquired β-lactamases displaying elevated expression of AmpC (14 isolates), elevated expression of efflux pumps (11 isolates), and/or decrease or loss of OprD (22 isolates) were susceptible to ceftolozane-tazobactam. Ceftolozane-tazobactam was active against >75% of the Enterobacterales isolates from the ASPECT-NP trial that did not carry carbapenemases. K. pneumoniae resistant to ceftolozane-tazobactam might represent a challenge for treatment due to its multiple resistance mechanisms. Ceftolozane-tazobactam was among the agents displaying the greatest activity against P. aeruginosa isolates.

Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa

“…The results showed that a large percentage of strains decreased their MIC to CZA and C/T in the presence of the efflux pump inhibitor, PAβN, but most of them were minor changes (RND 2x) and insufficient to increase the proportion of susceptible strains in this population. This may indicate that the effectiveness of the RND-mediated efflux of CZA and C/T is generally low compared with other antibiotics, consistent with previous reports ( Cabot et al, 2014 ; Castanheira et al, 2014 ; Castanheira et al, 2021 ). In fact, the changes observed at the population scale in susceptibility to CZA due to the presence of PAβN were not significant, opposite to C/T, for which these changes were significant.…”

“…These highlights the need to apply a modified intrinsic resistance concept that can be advantageous when clinical isolates are studied, named here as “baseline susceptibility”. It has been reported that modification and/or overexpression of the RND efflux pumps are implicated in acquired resistance to CZA and C/T in Non-CP-CRPA, usually in combination with other mechanisms of beta-lactam resistance such as the oprD decrease expression, PBPs modification or ampC overexpression ( Castanheira et al, 2014 ; Wi et al, 2018 ; Castanheira et al, 2021 ; Gomis-Font et al, 2021 ). In particular, the overexpression and/or structural modification of MexAB-OprM is the most common RND-mediated mechanism observed in acquired resistance to CZA and C/T ( Castanheira et al, 2014 ; Wi et al, 2018 ; Gomis-Font et al, 2021 ).…”

“…Regarding C/T, studies support a higher affinity of ceftolozane for certain PBPs than other beta-lactams, and C/T activity is less affected by changes in efflux pumps and porin permeability, hindering the emergence of C/T resistance mechanisms ( Cabot et al, 2014 ; Wi et al, 2018 ; Martin-Loeches et al, 2020 ). However, over-expression of MexAB-OprM and/or structural modification of MexCD-OprJ have been directly implicated in decreased susceptibility to C/T ( Castanheira et al, 2021 ; Gomis-Font et al, 2021 ).…”

Ceftolozane-tazobactam activity against clinical isolates of Pseudomonas aeruginosa from ICU patients with pneumonia: United States, 2015–2018

“…One limitation of this study is that molecular characterization of the resistant isolates was not performed. There have been recent publications describing the mechanisms of ceftolozanetazobactam resistance in P. aeruginosa , which include the acquisition of extended-spectrum or metallo-β-lactamases, mutations in chromosomal AmpC ( Pseudomonas -derived cephalosporinase), and/or mutations in the outer membrane porin, OprD ( Fraile-Ribot et al, 2018 ;Castanheira et al, 2021 ;Simner et al, 2021 ).…”

Detection of Klebsiella pneumoniae DNA by PCR-Based CRISPR-lbCas12a System

“…The golden standard of nucleic acid detection is qPCR. qPCR specifically and accurately identifies K. pneumoniae from bacteria like E. coli and S. aureus [19] and its antibiotic-resistant genes, such as ESBL-encoding and Fosfomycin-encoding genes [20]. However, qPCR needs expensive equipment and skilled workers [21].…”