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

Abstract

Introduction: 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.

Methods: Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla _PER 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 bla _PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics.

Results: Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla _PER. One isolate carried bla _PER but was CZA-susceptible. 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 bla _PER-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 bla _PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the in vitro evolved isolate.

Discussion: PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with bla_PER-3 gene implicated in resistance to CZA and other β-lactams.

Keywords: PER-3 extended-spectrum β-lactamase; Pseudomonas aeruginosa; blaPER-3 gene; ceftazidime/avibactam resistance; mutations conferring CZA resistance.

Figure 1

Genetic characteristics of PER-positive and PER-negative isolates. All PER-positive isolates except 1368 had an ISCR1 element upstream blaPER-3 gene composed by the ISCR1 gene coding for a site-specific recombinase and two adjacent 28bp-target sequences (vertical bars). *detected only in isolate 1673. Striped bars represent sequences that are non-homologous among isolates. CZA, ceftazidime-avibactam; C/T, ceftolozane-tazobactam.

Figure 2

Genomic regions involved in loss of bla_PER-3 gene in isolate MF-1 and in vitro evolved isolate MF-2. (A) MF-1 chromosome was obtained through hybrid alignment of long+short reads. Coverage of MF-2 short reads mapped against MF-1 chromosome is shown. The green square indicates the uncovered region. (B) The genetic content of the 46,933 bp deletion is shown. The bla _PER-3 gene is embedded in a class 1 integron (red lines). The 3`CS (conserved segment) of the integron is duplicated as 3`CS-1 and 3`CS-2 and the 5`CS includes the class 1 integrase (blue lines). Purple and pink curved lines show the upstream and downstream limits of the deletion. Black vertical bars: 28 bp-target sequences of ISCR1.