RESUMO
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 ß-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 ß-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 ß-lactamsin the in vitro evolved isolate. Discussion: PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other ß-lactams.
Assuntos
Antibacterianos , Compostos Azabicíclicos , Proteínas de Bactérias , Ceftazidima , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas , Pseudomonas aeruginosa , beta-Lactamases , Ceftazidima/farmacologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Compostos Azabicíclicos/farmacologia , Humanos , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Infecções por Pseudomonas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Chile , Sequenciamento Completo do Genoma , MutaçãoRESUMO
A ceftazidime-avibactam-resistant KPC-producing Pseudomonas aeruginosa strain was isolated in Argentina from a tracheal aspirate. The patient was treated with ceftazidime-avibactam in combination with other agents for 130 days. Whole-genome sequencing of P. aeruginosa identified a D179Y substitution in the Ω loop of KPC-3, corresponding to KPC-31, integrated at the chromosome. The strain belonged to the sequence type 235/O11 (ST235/O11) high-risk clone. Evaluation of carbapenemase detection assays most used by clinical laboratories failed to identify the isolate as a KPC producer.
Assuntos
Klebsiella pneumoniae , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ceftazidima/farmacologia , Ceftazidima/uso terapêutico , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , beta-Lactamases/genética , Combinação de Medicamentos , Proteínas de Bactérias/genéticaRESUMO
Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing Pseudomonas aeruginosa (KPC-PA) and Enterobacterales. CAZ/AVI resistance is driven by several mechanisms. In P. aeruginosa this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in Enterobacterales it frequently occurs through D179Y substitution in the active site of KPC enzyme. This aminoacid change abolishes AVI binding to the KPC active site, hence inhibition is impaired. However, this substitution also decreases KPC-mediated resistance to carbapenems ("see-saw" effect). The goal of this work was to characterize the in vivo acquisition of CAZ/AVI resistance through D179Y substitution in a KPC-PA isolated from a hospitalized patient after CAZ/AVI treatment. Two KPC-PA isolates were obtained. The first isolate, PA-1, was obtained before CAZ/AVI treatment and was susceptible to CAZ/AVI. The second isolate, PA-2, was obtained after CAZ/AVI treatment and exhibited high-level CAZ/AVI resistance. Characterization of isolates PA-1 and PA-2 was performed through short and long-read whole genome sequencing analysis. The hybrid assembly showed that PA-1 and PA-2A had a single plasmid of 54,030 bp, named pPA-1 and pPA-2 respectively. Each plasmid harbored two copies of the bla KPC-containing Tn4401b transposon. However, while pPA-1 carried two copies of bla KPC-2, pPA-2 had one copy of bla KPC-2 and one copy of bla KPC-33, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the "see-saw" effect. The bla KPC-33 allele was detected only through hybrid assembly using a long-read-first approach. The present work describes a KPC-PA isolate harboring a plasmid-borne CAZ/AVI resistance mechanism based on two copies of bla KPC-2-Tn4401b and D179Y mutation in one of them, that is not associated with loss of resistance to carbapenems. These findings highlight the usefulness of a fine-tuned combined analysis of short and long-read data to detect similar emerging resistance mechanisms.
Assuntos
Ceftazidima , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos , Carbapenêmicos/farmacologia , Ceftazidima/farmacologia , Combinação de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Mutação , Porinas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
A rapid colorimetric method, the Andrade screening antimicrobial test, was compared with the E-test method to detect ceftazidime/avibactam (CZA) resistance in carbapenem resistant Enterobacterales clinical isolates. A 106 non-duplicated isolates (86 susceptible and 20 resistant to CZA) were chosen for validation. The sensitivity and specificity were 100%. This method investigates CZA resistance regardless of the resistance mechanism involved. It represents an economical and easy technique that can be applied to routine microbiology laboratories. It allows the detection of CZA resistance at 3 hours of incubation and consequently, the early implementation of accurate therapeutic interventions.