Browsing by Author "Wozniak, Aniela"
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Item A multispecies outbreak of carbapenem-resistant bacteria harboring the blaKPC gene in a non-classical transposon element(2021) Wozniak, Aniela; Figueroa, Cristian; Moya-Flores, Francisco; Guggiana, Piero; Castillo, Claudia; Rivas, Lina; Munita, José; García, Patricia C.Klebsiella pneumoniae is the most frequent KPC-producing bacteria. The blaKPC gene is frequently embedded in Tn4401 transposon, and less frequently in non-Tn4401 elements (NTEKPC) variants I-III. The first case of KPC in the UC-CHRISTUS Clinical Hospital was detected in Pseudomonas aeruginosa. Soon after this event, KPC was detected in 2 additional Pseudomonas aeruginosa, 3 Escherichia coli, 3 Enterobacter cloacae, 3 Klebsiella pneumoniae, and 1 Citrobacter freundii, isolated from 6 different patients. We aimed to elucidate the possible mechanisms of genetic transfer and dissemination of the blaKPC gene among isolates of this multispecies outbreak. A molecular epidemiology analysis of the above mentioned clinical isolates (n = 13) through Multi-Locus Sequence Typing, plasmid analysis, Pulsed-Field Gel-Electrophoresis, and Whole-genome sequencing (WGS) was performed.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.Item Catheter-associated bloodstream infection caused by Leifsonia aquatica in a haemodialysis patient: a case report(Microbiology Society, 2012) Porte, Lorena; Soto, Andres; Andrighetti, Daniela; Dabanch, Jeannette; Braun, Stephanie; Saldivia, Alejandra; Flores, Juan Carlos; Wozniak, Aniela; Garcia, Patricia; Weitzel, ThomasLeifsonia aquatica is an aquatic coryneform rod that is capable of forming biofilms in environmental water sources. It has rarely been associated with human infections and its pathogenicity and clinical significance are uncertain. We describe a case of catheter-related bloodstream infection in a haemodialysis patient. The isolate grew on conventional media as a yellow-pigmented colony, but identification required molecular methods. Although the strain displayed reduced sensitivity to vancomycin, the clinical outcome was favourable after catheter removal and intravenous treatment with this antibiotic. Our report gives further evidence of the capability of this aquatic bacterium to cause human infection.Publication Chromosome-Mediated Colistin Resistance in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli: Mutation Analysis in the Light of Genetic Background(2023) Riquelme, María; Martinez, Rodrigo; Brito, Bárbara; García, Patricia; Legarraga, Paulette; Wozniak, AnielaPurpose: Colistin resistance mechanisms involving mutations in chromosomal genes associated with LPS modification are not completely understood. Mutations in genes coding for the MgrB regulator frequently account for colistin resistance in Klebsiella pneumoniae, whereas mutations in genes coding for PhoPQ and PmrAB are frequent in E. coli. Our aim was to perform a genetic analysis of chromosomal mutations in colistin-resistant (MIC ≥4 µg/mL) clinical isolates of K. pneumoniae (n = 8) and E. coli (n = 7) of different STs. Methods: Isolates were obtained in a 3-year period in a university hospital in Santiago, Chile. Susceptibility to colistin, aminoglycosides, cephalosporins, carbapenems and ciprofloxacin was determined through broth microdilution. Whole genome sequencing was performed for all isolates and chromosomal gene sequences were compared with sequences of colistin-susceptible isolates of the same sequence types. Results: None of the isolates carried mcr genes. Most of the isolates were susceptible to all the antibiotics analyzed. E. coli isolates were ST69, ST127, ST59, ST131 and ST14, and K. pneumoniae isolates were ST454, ST45, ST6293, ST380 and ST25. All the isolates had mutations in chromosomal genes analyzed. K. pneumoniae had mutations mainly in mgrB gene, whereas E. coli had mutations in pmrA, pmrB and pmrE genes. Most of the amino acid changes in LPS-modifying enzymes of colistin-resistant isolates were found in colistin-susceptible isolates of the same and/or different ST. Eleven of them were found only in colistin-resistant isolates. Conclusion: Colistin resistance mechanisms depend on genetic background, and are due to chromosomal mutations, which implies a lower risk of transmission than plasmid-mediated genes. Colistin resistance is not associated with multidrug-resistance, nor to high-risk sequence types.Item Real-world Performance of Susceptibility Testing for Ceftolozane/Tazobactam Against Non-Carbapenemase-Producing Carbapenem-Resistant Pseudomonas aeruginosa(2021) Rivas, Lina; Alcalde-Rico, Manuel; Martínez, José R.; Moreno, Victoria; Rojas, Pamela; Wozniak, Aniela; García, Patricia; Olivares, Jorge; Miller, William R.; Arias, Cesar A.; Khan, Ayesha; Munita, JoséCeftolozane/tazbactam (C/T) is a potent anti-pseudomonal agent that has clinical utility against infections caused by non-carbapenemase producing carbapenem-resistant P. aeruginosa (non-CP-CR-PA). Accurate, precise and reliable antimicrobial susceptibility testing (AST) is crucial to guide clinical decisions. However, studies assessing the performance of different AST methods against non-CP-CR-PA- (the main clinical niche for C/T), are lacking. Here, we evaluated performance of gradient strips (Etest and MIC test strip (MTS), and disk diffusion (DD) using CLSI breakpoints. Additionally, we assessed the performance of DD using EUCAST breakpoints. For all susceptibility tests, we used a collection of 97 non-CP-CR-PA clinical isolates recovered from 11 Chilean hospitals. Both gradient strips and DD had acceptable performance when using CLSI breakpoints, yielding a categorical agreement (CA) of >90% and 92%, respectively. In contrast, DD using EUCAST breakpoints performed sub-optimally (CA 81%). MTS yielded a higher essential agreement (EA, >90%) than Etest (84%). Importantly, the performance of all methods varied significantly when the isolates were stratified by their degree of susceptibility to other anti-pseudomonal β-lactams. All methods had 100% CA when testing isolates that were pan-susceptible to all β-lactams (Pan-β-S). However, the CA markedly decreased when testing isolates resistant to all β-lactams (Pan-β-R). Indeed, the CA was 81% for Etest (6 errors), 78% for MTS (7 errors) and 78% and 56% for DD when using CLSI (7 errors) or EUCAST breakpoints (14 errors), respectively. Our results suggest that all manual AST methods have strikingly decreased performance in the context of Pan-β-R P. aeruginosa with potentially major clinical implications.Publication Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa(2022) Contreras-Gómez, María José; Martínez, José Rodrigo Waldemar; Rivas Jiménez, Lina María; Riquelme-Neira, Roberto; Ugalde, Juan A.; Wozniak, Aniela; García, Patricia; Munita, Jose M.; Olivares-Pacheco, Jorge; Alcalde-Rico, ManuelCarbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine β-naphthylamide (PAβN). In the absence of PAβN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAβN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAβN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.