Browsing by Author "Olivares-Pacheco, J."

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    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, P.; Brito, B.; Alcalde-Rico, M.; Munita, J.M.; Martínez, J.R.W.; Olivares-Pacheco, J.; Quiroz, V.; Wozniak, A.
    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 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.
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    High prevalence of class 1 integrase and characterization of class 1 integron gene cassettes in multiresistant bacteria isolated from the gut microbiota of extended antibiotic treated Salmo salar fish farms
    (2019) Vásquez-Ponce, F.; Higuera-Llantén, S.; Cortés, J.; Zimin-Veselkoff, N.; Marshall, S.H.; Mardones, F.O.; Olivares-Pacheco, J.
    The use of antimicrobials in aquaculture is a common practice. Chile is second larger producer of salmon worldwide, but unfortunately is the first consumer of antibiotics. Tonnes of florfenicol and oxytetracycline yearly are used in the Chilean salmoniculture to control the pathogens that threaten the sustainability of the industry. This excessive use of antibiotics have selected populations of resistant bacteria from the sediments and the water column that sorround the fish farms. In a recent work, our lab described the high prevalence of multiresistant bacteria and Antibiotic Resistance Genes (ARGs) in the gut microbiota of Antlactic salmon (Salmo salar) treated with high doses of antibiotics. In this work, we revisited the analysis of the previously described gut multiresistant bacteria grouped in banks of florfenicol resistant isolates (FB) and oxytetracycline resistant isolates (OB) looking for the presence of integron-integrase elements. These elements have been described as an important players in the Antimicrobial Resistance (AMR) phenomenon and they are considered a good markers of the anthopogenic activities pollution. The results showed that the 100% of the multiresistant isolates present the class 1 intagrase. Despite this result, no isolate from FB showed the typical structure of class 1 integrons: the presence in 3’-CS of qacEΔ1/sul1 genes. While in OB, only 23% of the isolates showed this characteristic structure. Additionally, only four isolates of OB and none of FB showed recognisable gene cassettes and no genes of resistance to florfenicol and oxytetacycline appeared in them. Of these four isolates, three of them showed a single gene cassette containing the dfrA-14 gene, which confers resistance to trimethoprim. Whilst the other isolate showed the aac(6’)31-qacH-blaoxa2 genes, which confers resistance to aminoglycosides, quaternary ammonium compounds and beta-lactams, respectively. Finally, it was possible to demonstrate that the described integrons probably come from anthropogenic activities like clinical settings and/or industrial animal husbandry, since they show integrases proteins identical to those carried by human pathogens.