Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa Provisionally Accepted

Katherine D. Soto^{1, 2} Manuel Alcalde-Rico^{2, 3, 4, 5} Juan A. Ugalde^{2, 6} Jorge A. Olivares Pacheco^{2, 3} Valeria Quiroz^{1, 2} Bárbara Brito⁷ Lina M. Rivas^{2, 8} José M. Munita^{2, 9} Patricia García^{1, 2, 10} Aniela Wozniak^{1, 2, 10*}

• ¹Pontificia Universidad Católica de Chile, Chile
• ²Millenium Nucleus for Collaborative Research on Bacterial Resistance (MICROB-R), Chile
• ³Pontificia Universidad Católica de Valparaíso, Chile
• ⁴Departamento de Fisiología Médica y Biofísica, Instituto de Biomedicina de Sevilla (IBIS), Spain
• ⁵Carlos III Health Institute (ISCIII), Spain
• ⁶Faculty of Life Sciences, Andres Bello University, Chile
• ⁷University of Technology Sydney, Australia
• ⁸Facultad de Medicina Clinica Alemana, Universidad del Desarrollo, Chile
• ⁹Clínica Alemana, Universidad del Desarrollo, Chile
• ¹⁰Red de Salud UC-Christus, Chile

Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum β-lactamase, has been well documented in vitro.However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while blaPER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin.Loss of blaPER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried blaPER. One isolate carried blaPER but was CZAsusceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their blaPER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum β-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of blaPER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the in vitro evolved isolate. PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other β-lactams.