Browsing by Author "Quiroz, Valeria"
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Publication Acquisition of resistance to ceftazidime-avibactam during infection treatment in Pseudomonas aeruginosa through D179Y mutation in one of two blaKPC-2 gene copies without losing carbapenem resistance(2022) García, Patricia; Brito, Bárbara; Alcalde-Rico, Manuel; Munita, José; Martínez, José R.; Olivares Pacheco, Jorge; Quiroz, Valeria; Wozniak, AnielaCeftazidime/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 blaKPC-containing Tn4401b transposon. However, while pPA-1 carried two copies of blaKPC-2, pPA-2 had one copy of blaKPC-2 and one copy of blaKPC-33, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the “see-saw” effect. The blaKPC-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 blaKPC-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.Publication Actividad in vitro de ceftazidima/avibactam contra aislados clínicos de Klebsiella pneumoniae resistente a carbapenémicos y comparación de técnicas de susceptibilidad(2025) Quiroz, Valeria; Rivas, Lina María; Soto, Katherine; Sanfurgo, Sanfurgo; Antolínez, Yohanna; Tenner, Rebecca; Araos, Rafael; Díaz, Lorena; García, Patricia; Munita, JoséIntroducción: Klebsiella pneumoniae resistente a carbapenémicos constituye un reto clínico debido a la limitada disponibilidad de opciones terapéuticas. La resistencia a carbapenémicos puede darse con producción o ausencia de carbapenemasas; en ambos casos ceftazidima/avibactam es una alternativa terapéutica prometedora. Objetivos: Evaluar la actividad in vitro de ceftazidima/avibactan sobre ceps de Klebsiella pneumoniae resistentes a carbapenemasas y comparar diferentes técnicas de medición de su actividad antimicrobiana. Métodos: La actividad in vitro de ceftazidima/avibactam fue evaluada en 186 aislados clínicos de K. pneumoniae resistente a carbapenémicos provenientes de 11 hospitales de Chile; 59 productores de blaKPC y 127 no productores de carbapenemasas. Se realizó determinación de concentración inhibitoria mínima (CIM) por la técnica de microdilución en caldo y fue comparada con epsilometría y disco difusión. Resultados: 100% de los aislados de K. pneumoniae resistente a carbapenémicos fueron susceptibles a ceftazidima/avibactam con CIM entre 0,25 y 8 μg/mL. Quince aislados (12 de ellos no productores de carbapenemasas) mostraron CIM de 8 μg/mL. Se observó alta concordancia (> 96%) comparando la microdilución en caldo con la epsilometría y disco difusión. Discusión: ceftazidima/avibactam se mostró activo en aislados clínicos de K. pneumoniae resistente a carbapenémicos chilenos. Debido a que se observaron aislados con CIM cercana al punto de corte, se recomienda vigilar su actividad. El rendimiento de la epsilometría y la disco difusión mostró una concordancia aceptable frente a la microdilución en caldo, por lo que se sugieren como alternativas en la práctica clínica.Publication Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in Chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa(2024) Soto, Katherine; Alcalde, Manuel; Ugalde, Juan; Olivares, Jorge; Quiroz, Valeria; Brito, Bárbara; Rivas Jiménez, Lina María; Munita, Jose M.; García, Patricia; Wozniak, AnielaIntroduction: 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 blaPER-3 gene implicated in resistance to CZA and other β-lactams.Publication Multispecies emergence of dual blaKPC/NDM carbapenemase-producing Enterobacterales recovered from invasive infections in Chile(2025) Quesille, Ana; Solar, Camila; Martínez, Jose; Rivas Jiménez, Lina María; Quiroz, Valeria; González, Ana; Riquelme, Roberto; Ugalde, Juan; Peters, Anne; Ortega, Oscar; Araos Bralic, Rafael Ignacio; García, Patricia; Lebreton, Francois; Munita, Jose M.; Diaz, LorenaCarbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) represent a significant global threat. The emergence of dual CP-CRE is particularly alarming, as they can potentially compromise the efficacy of newer antibiotics, further decreasing therapeutic alternatives. Herein, we report the emergence of multiple species of CP-CRE recovered from invasive infections in Chile that simultaneously harbor blaKPC and blaNDM and provide an in-depth genomic characterization of these worrisome pathogens. We collected carbapenem-resistant Enterobacterales (CRE) isolates from invasive infections over a 4-year period, across 11 healthcare centers in Chile. Bacterial species and the presence of carbapenemase genes were confirmed using MALDI-TOF and PCR assays, respectively. Antimicrobial susceptibility testing was conducted through disk diffusion and broth microdilution methods. Dual CP-CRE isolates were subjected to short- and long-read whole genome sequencing to perform a detailed genomic characterization of the isolates and of the mobile genetic elements harboring the enzymes. From a total of 1,335 CRE isolates, we observed an increase in the prevalence of CP-CRE, from 11% in 2019 to 38% in 2022. A total of 11 dual CP-CRE isolates were recovered, all of them harboring blaKPC and blaNDM. Species corresponded to Escherichia coli (n = 6), Klebsiella pneumoniae (n = 2), Klebsiella oxytoca (n = 2), and Citrobacter freundii (n = 1). Dual CP-CRE isolates exhibited resistance to all tested β-lactams except for cefiderocol. The blaKPC and blaNDM encoding genes were located on independent plasmids. Platforms harboring blaKPC were diverse and included IncN, IncF, and IncFIB plasmids. In contrast, blaNDM-7 was only found on fairly conserved IncX3 plasmids. We report that a rapid increase of CP-CRE in Chile, alongside with the emergence of multiple bacterial species of CP-CRE co-harboring blaKPC-2/3 and blaNDM-7, underscores a critical public health challenge. Our data suggest that the dissemination of blaNDM-7 was predominantly facilitated by IncX3 plasmids, whereas the spread of blaKPC involved multiple plasmid backbones. Active surveillance and genomic monitoring are critical to inform public policy and curtail the spread of these highly resistant pathogens.